EP0510064A1 - Procedes et appareils de detection du dichroisme circulaire et de l'absorption spectrophotometrique - Google Patents

Procedes et appareils de detection du dichroisme circulaire et de l'absorption spectrophotometrique

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Publication number
EP0510064A1
EP0510064A1 EP91902458A EP91902458A EP0510064A1 EP 0510064 A1 EP0510064 A1 EP 0510064A1 EP 91902458 A EP91902458 A EP 91902458A EP 91902458 A EP91902458 A EP 91902458A EP 0510064 A1 EP0510064 A1 EP 0510064A1
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EP
European Patent Office
Prior art keywords
test sample
cholesterol
determining
detection method
recited
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EP91902458A
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German (de)
English (en)
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EP0510064A4 (en
Inventor
Neil Purdie
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Research Corp Technologies Inc
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Research Corp Technologies Inc
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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/743—Steroid hormones
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/19—Dichroism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour

Definitions

  • the present invention is concerned with the use of circular dichroism and absorption detection in clinical chemistry detection methods. More specifically, with their use in the measurement of cholesterol levels and direct measurement of cholesterol subfractions in clinical samples, as well as in the detection of anabolic steroids and other steroid products, and in the measurement of Hpoprotein levels in a serum test sample.
  • the invention is also concerned with providing certain CD and absorption apparatuses useful in each of the aforesaid chemical methods.
  • Spectrophotometric absorption refers to the measurement of the absorption or transmission of incident light through solutions of test compounds.
  • compounds of interest have characteristic absorption spectra, transmitting or absorbing specific wavelengths of light, which can be used to determine the presence of these compounds in test samples.
  • Instruments designed for spectrophotometric absorption have a light source, for which the emitted wavelength is known and may be adjusted, and one or more detectors sensitive to desired wavelengths of transmitted light.
  • Spectrophotometric absorption can be used to determine the amounts of a given compound that are present in a test sample.
  • Circular dichroism is a special type of absorption method in which the molecular composition of the compound results in differential absorption of incident light not only at a specific wavelength but also of a particular polarization state.
  • Circular dichroism is a chiroptical method which allows one to differentiate between different enantiomers, that is, optical isomers having one or more asymmetric carbon atom (chiral) centers.
  • CD generally a sample is illuminated by two circularly polarized beams of light traveling in unison. Both beams pass through the sample simultaneously and are absorbed. If the sample is optically active, the beams are absorbed to a different extent. The differences in absorption of the beams can then be displayed as a function of the wavelength of the incident light beam as a CD spectrum.
  • CD has been fully described in scientific literature (1) .
  • Early applications of the CD method primarily dealt with elucidation of molecular structures, especially natural products for. which a technique capable of confirming or establishing absolute stereochemistry was critical.
  • CD has also reportedly been used in a clinical method to quantitatively determine unconjugated bilirubin in blood plasma (2) .
  • a complex was formed between bilirubin and human serum albumin as a CD probe for bilirubin analysis.
  • HDL-C high density Hpoprotein
  • LDL-C low density Hpoprotein
  • VLDL-C very low density Hpoprotein cholesterol
  • LDL-C are to be avoided where possible.
  • HDL-C is beneficial, provided the level is not excessively low, less than 30mg/dL (7) .
  • VLDL-C cholesterol has not been implicated in any risk determination, but high triglyceride itself can be a serious problem.
  • ' total and HDL-C cholesterols are measured directly.
  • VLDL-C is taken to be a fixed fraction (e.g, 0.2) of the triglyceride, which is also measured directly in a separate assay.
  • LDL-C is calculated from these figures and is not measured directly. The propagation of errors in each of the three independent measurements makes LDL-C the fraction known with least overall accuracy, although it may be the most significant aspect of cardio-vascular risk. Because of this, it is difficult to meaningfully monitor and justify that clinical progress has been made in LDL-C reduction therapy with time.
  • ketosteroids are amenable to direct CD detection (11) .
  • An object of the present invention is to provide spectrophotometric methods for direct measurement of cholesterol in clinical samples, as it exists in association with several particular Hpoprotein sub- fractions. These spectrophotometric methods encompass both CD and conventional absorption spectrophotometry, either separately or in combination.
  • the CD methods permit measurement of anabolic steroids or other steroid products as well.
  • Another object of the present invention is to provide a method of measuring cholesterol levels in a clinical test sample, wherein the combined LDL-C + VLDL-C level is determined directly, or where LDL-C and VLDL-C levels separately can be directly determined, using either CD or spectrophotometric absorption. It is a further object of the present invention to provide a method wherein LDL-C, VLDL-C, combined LDL-C + VLDL-C and HDL-
  • C levels in a test sample can all be determined directly, and simultaneously, if desired. It is still a further object of the invention to combine direct measurement of cholesterol subfractions by CD absorption with the direct measurement of TC by spectrophotometric absorption, while using identical reaction conditions. It is also an object of the present invention to provide novel apparatuses to carry out such detection methods. Another object of the present invention is to provide methods and apparatuses for detecting the presence of lipoproteins which are associated with different cholesterol subfractions.
  • the present invention provides for a clinical method for determining the amount of cholesterol, Hpoprotein, anabolic steroid or other steroidal product in a serum test sample, by forming a reaction product with the cholesterol, Hpoprotein anabolic steroid or other steroidal product in the test sample, and then either perform step (a), (a') or (a") :
  • Step (a) determining the CD and/or absorption of the test sample over a range from about 150 to 700 nm
  • Step (a 1 ) determining the CD absorption of the test sample at one or more discrete wavelengths within a range from about 150 to 700 nm (preferably from about 240 nm to 625 nm) ;
  • Step (a) determining the spectrophotometric ab- sorption spectrum of the test sample at one or more discrete wavelengths within a range from about 400 nm to 700 nm (preferably about 450 to 625 nm) .
  • a detection instrument for determining the amount of VLDL-C + LDL-C, HDL-C and total cholesterol (TC) present in a test sample including means for determining the amount of HDL-C present in the sample by CD absorption at a first wavelength or a first and a second wavelength, means for determining the amount of VLDL-C + LDL-C in the sample by CD absorption at a third wavelength, and means for determining the amount of TC in the sample by spectrophotometric absorption at the third wavelength, or alternatively, means for determining the amount of TC in the sample by calculation or computation, based upon values obtained for VLDL-C + LDL-C and HDL-C in the sample.
  • a detection instrument for determining the amount of VLDL-C + LDL-C, HDL-C and total cholesterol (TC) present in a sample including means for determining the amount of TC in the sample by spectrophotometric absorption at a first wavelength, means for determining the amount of VLDL-C + LDL-C in the sample by CD absorption at the first wavelength, and means for determining the amount of HDL-C in the sample by calculation or computation, based upon values obtained for VLDL-C + LDL-C and TC in the sample.
  • a spectrophotometric absorption instrument for determining the amount of total cholesterol (TC) , combined VLDL-C + LDL-C, and HDL-C present in a test sample, the instrument comprising spectrophotometric absorption means for directly determining the amount of TC in the sample at a first wavelength, spectrophoto ⁇ metric absorption means for directly determining HDL-C at a second wavelength, means for determining combined LDL-C + VLDL-C by computation using the values obtained for TC and HDL-C, and optionally spectrophotometric ab ⁇ sorption means for directly determining the amount of VLDL-C in the test sample.
  • CD instrument means a Circular Dichroism Instrument. Such instruments are available commercially or may be constructed from parts, which may be commercially available. Additionally, Figure 6 is included herewith to provide a simple schematic of how a CD works. As can be seen in Figure 6, light from a light source (LS) is linearly polarized with linear polarizers (P) and then circularly polarized in opposite directions by circular polarizers (Q) and then shown through a specimen cell (S) , whereupon absorbance is measured by a detector (D) , the difference is measured and plotted as a function of wavelength to produce a CD spectrum, or alternatively, may be recorded at preselected wavelengths.
  • LS light from a light source
  • P linear polarizers
  • Q circular polarizers
  • S specimen cell
  • D detector
  • LDL cholesterol means low density Hpoprotein cholesterol.
  • HDL cholesterol abbreviated HDL-C
  • VLDL cholesterol abbreviated VLDL-C
  • total cholesterol abbreviated TC
  • anabolic steroid means steroids such as testosterone and its 17-epimer, dehydrotestosterone, 17-alkyltestosterones , nortestosterone, mestanolone, methandriol and the like.
  • steroidal products means other steroids, such as 17 ketosteroids, adrenal corticoids and the like.
  • Hpoprotein as used herein, means macromolecular complexes of lipids and proteins found in human plasma. Exemplary of such lipoproteins are low density Hpoprotein, very low density Hpoprotein, intermediate density Hpoprotein, LP (a) lipoproteins, chylomicrons, apolipoproteins (A-l, A-ll, B-48, B-100, c, D, E, etc) , and the like.
  • Chugaev reaction product means the reaction product of cholesterol, an anabolic steroid or steroidal product or Hpoprotein with Chugaev reactants such as 20% w/v ZnCl 2 in glacial acetic acid and 98% acetyl chloride, or the like.
  • Chugaev Reaction utilized herein to form the Chugaev reaction products of the present invention, is disclosed in the literature (12) and is suggested to involve dehydration and opening of the B-ring of the steroid to form an optically active colored reaction product.
  • test sample refers to a whole blood test sample or a whole blood test sample having the cell bodies removed therefrom by centrifugal force or through the use of an appropriate filter mechanism, both of which means are well known to those skilled in the art.
  • bilirubin conjugate means the conjugate found between bilirubin and a serum Hpoprotein, apolipoprotein or protein at about a pH of 5.0-5.1.
  • the conjugate is formed with a Hpoprotein or apolipoprotein, which is associated with a cholesterol subfraction.
  • spectrophotometric absorption refers to measurement of the absorption (or, conversely, transmission) of incident light by colored compounds at specific wavelengths, irrespective of the state of polarization of the light.
  • alkali metal sulfate as used herein, means sodium sulfate, lithium sulfate, potassium sulfate, and the like, wherein a sulfate salt is formed with an alkali metal.
  • alkali earth metal sulfate means calcium sulfate, barium sulfate, and the like, wherein a sulfate salt is formed with an alkali earth metal.
  • transition metal sulfate as used herein, means scandium sulfate, titanium sulfate, chromium sulfate, manganese sulfate, nickel sulfate, zinc sulfate, copper sulfate, cadmium sulfate, and the like, wherein a sulfate salt is formed with a transition metal.
  • alkali metal perchlorate means sodium perchlorate, lithium perchlorate, potassium perchlorate, or the like, wherein a perchlorate salt is formed with an alkali metal.
  • Figure 1 is a full CD spectrum for the optically active colored product obtained from the reaction of Chugaev reagents with cholesterol.
  • Curve (a) is re- presentative of the total cholesterol, while the shaded area is the spectrum after the addition of the LDL precipitating agent and is therefore representative of the HDL fraction only.
  • Figure 6 is a schematic of a CD, wherein: LS is the high intensity conventional light source or laser source; Ml and M2 are monochromators required for full spectral data; P is the linearly polarizing element; Q is the circularly polarizing element; S is the sample cell; D is the detector (of which there may be up to three) ; and REC is the recorder.
  • Figure 7 is a graph that shows the normal absorption spectrum of the optically active colored product obtained from the reaction of Chugaev reagents with cholesterol, wherein:
  • Absorption Curve (b) is observed after reaction with the Chugaev reagent to which has been added approximately 2% w/v Na 2 S0 4 or other alkali metal or alkaline earth metal sulfate; and Absorption Curve ( ⁇ ) is observed after reaction with the Chugaev reagent to which has been added approximately 2% w/v dextran sulfate or alkali metal perchlorate such as sodium perchlorate.
  • the reagents utilized in the Chugaev reaction are 20% w/v ZnCl 2 in glacial acetic acid, and 98% acetyl chloride. They can be stored in separate containers and will remain stable for many weeks, when stored at about 40°C. Moreover, the degree of their dryness does not have to be carefully controlled.
  • the product of their reaction is a conjugated triene CD-active derivative of cholesterol which is reddish-orange in color. This is an improvement over presently known methods, wherein the colored species are secondary dyes and not cholesterol derivatives, and their intensities are only proportional to the original cholesterol concentration.
  • the reactants for the Chugaev reaction may also be stored together in a ratio of about a 1:1 to 4:1 ratio of ZnCl 2 in glacial acetic acid to 98% acetyl chloride, when stored under* airtight conditions in an amber glass, teflon or a similar container.
  • an extended period of stability against discoloration was observed for reactants stored together at 40°C in amber bottles for at least 4 weeks.
  • acetyl chloride is critical to making the Chugaev color reaction proceed in a reasonably short period of time ( « 8 min.)
  • the upper volume of acetyl chloride used is not as critical as the lower volume used.
  • the amount of acetyl chloride must be greater than 0.5 ml per 2.0 ml aliquot of the zinc chloride; spectral data have been obtained which are essentially the same when either 0.75 ml or 1.0 ml of acetyl chloride was mixed with a 2ml aliquot of the zinc chloride.
  • agents such as Na 2 S0 4 may be added to the reactant solutions in an amount of about 1-2% w/v, in order to dry the solutions out (remove water) and ,r stabilize the same.
  • the addition of the Na 2 S0 4 changes the CD and absorption curves obtained for the sample. Specifically, the CD and absorption curves shift and change so that over the range of about 240 to 625 nm, a single CD peak for HDL-C occurs at about 475- 480 nm.
  • the amount of HDL-C can be calculated from a negative peak occurring at about 390 nm and/or a positive peak at about 475 nm, or preferably the algebraic sum of the two peaks.
  • a second advantage of the present invention is its use of circular dichroism in a detection method for cholesterol, since CD allows for greater specificity and greater selectivity with respect to the different cho ⁇ lesterol subfractions than to spectrophotometric methods previously known in the art.
  • CD a specimen is illuminated by two circularly polarized beams of light, which are travelling in unison and are polarized in opposite direction. Both beams pass through the specimen simultaneously and are absorbed. If the specimen is optically active, the beams are absorbed to different extents. The differences are displayed as a function of the wavelength of the incident light beam as a CD spectrum. No difference is observed for optically inactive absorbers so these are not detected.
  • the technique is fully described in the literature (1) as are typical CD apparatuses.
  • the full CD spectrum for the orange colored optically active product from the Chugaev reaction with cholesterol is shown in Figure 1.
  • the sample is a chloroform solution of the NBS Cholesterol Standard Reference Material (SRM911a) .
  • SRM911a NBS Cholesterol Standard Reference Material
  • Exemplary of the advantages to using the Chugaev reaction with CD detection over previously known spectrophotometric absorption methods include the following: (i) the CD spectra are the same whether the cholesterol is present in the test sample as the free sterol or as a fatty acid ester, so enzymatic saponification of the ester is an unnecessary step; (ii) there is no interference from hemolyzed blood cells because the red pigments are not optically active and are therefore transparent to the CD detector;
  • the reference spectrum is measured for a primary standard material, namely the purest form of cholesterol available, and not for a secondary calibrator standard;
  • the color is very stable because in CD detection an absorbance difference is measured, so even if the color loses intensity with time, the difference remains virtually constant;
  • the HDL-C and the (VLDL+LDL)-C fractions are associated with different bands in the CD absorption spectrum and can be measured directly from the same specimen, Figure 1, without the need for a precipitation step to determine HDL-C.
  • measurements at 525 nm give results for the combined (VLDL+LDL)-C fractions and measurements at 390 nm (or preferably the algebraic sum of the negative and positive CD absorption peaks at 390 nm and 475 nm, respectively) give results for the HDL-C fraction.
  • band assignments were made by comparing CD spectrum for the total cholesterol, curve (a) in Figure 1, with the spectrum*for the same sample after the selective precipitation of the low density lipid fractions with phosphotungstate-Mg, i.e., the shaded area in Figure 1.
  • the 525 nm band maximum was calibrated using NBS cholesterol (SRM 911a) .
  • Calibration of the 390 nm maximum was done using secondary HDL-C calibrators supplied by Sigma Chemical Co.
  • the reagents can be added either in the order indicated in (a) Calibration of the Instrument; however, they can also be added simul ⁇ taneously as a premixed solution or they can be added in the reverse order, e.g. add the acetyl chloride first, followed by the ZnCl 2 reagent.
  • the latter mode of reagent addition had the unexpected effect of reducing the amount of precipitation in the test sample, thereby greatly reducing the scattering of incident light and thereby simplifying the subsequent measurement of absorption either by CD or by conventional spectroscopic absorption.
  • the optically active colored product of the Chugaev reactions with cholesterol in the test samples has an absorption spectrum that extends over the range of about 240-700 nm [Fig* 7, Absorption Curve (a)]. It shows a strong absorption maximum at about 525 nm, which is associated with and proportional to the total cholesterol (TC) in the sample.
  • TC total cholesterol
  • absorbance measurements at 525 nm can be used to determine TC. Calibration data from measurements at 525 nm suggest a molar absorptivity for the colored product to be on the order of about 13,500.
  • Transitional metal sulfates also showed this effect, however they formed colored solutions and, for that reason, are not the preferred choice.
  • Comparisons between the CD spectral data of clinical samples and commercial preparations available from Sigma Biochemical suggest that this 480 nm peak correlates with HDL-C.
  • Figures suggest a molar absorptivity for the 480 nm peak to be on the order of about 4,000.
  • the difference bet ⁇ ween the optical densities, measured at 525 nm is proportional to the sum of the combined VLDL-C + LDL-C subfractions.
  • the intensity of the band attributed to HDL-C is on the order of one half the intensity of the TC spectrum at about 480 nm, it is conceivable that a mathematical algorithm can be written to curve-fit the spectrum for total cholesterol obtained from the basic Chugaev reagents (between about 400-700 nm) with weighted averages of the spectra for the three subfractions. As such, it may be possible to carry out the cholesterol lipid analysis to be done using only the spectrum from the colored product of the reaction of cholesterol in the clinical sample with the basic Chugaev test reagent.
  • spectrophotometric absorption reactions do not require the use of a CD instrument, yet they offer similar opportunity for simultaneous, on ⁇ line detection of cholesterol and cholesterol subfractions in clinical samples.
  • the use of spectrophotometric absorption methods using such Chugaev reaction reagents also permits much greater sensitivity than the CD methods herein disclosed allow for, since only a very small portion of the incident light can be used for CD signal generation.
  • the spectrophotometric absorption methods herein disclosed permit the use of smaller volumes of sample, thereby reducing possible interferences caused by other materials and the total amount of precipitates formed by the reaction. Conversely, however, these reactions are more susceptible than CD to interferences from pigments released by hemolysis of the blood samples.
  • a novel spectrophotometric absorption detection method wherein reagents are reacted with cholesterol in clinical samples so that a direct measurement of cholesterol subfractions can be made.
  • the measurements can be made either as a full spectrum over the range of about 400-700 nm or at two or more selected wavelengths, namely about 480 nm for HDL-C, 500 nm for VLDL-C, and 525 nm for combined VLDL-C + LDL-C (or TC, as desired) .
  • the major procedural difference between the absorption and the CD method relates to the standards used.
  • lipoproteins associated with the VLDL- C + LDL-C fraction are usually designated beta lipo ⁇ proteins and include B-100 C and E apliproproteins, while lipoproteins associated with the HDL-C fraction are designated alpha lipoproteins and include A-l, A- 11, C, D and E apoliproproteins.
  • the bilirubin conjugates which are formed with the HDL-C associated lipoproteins or apoliproproteins are measured directly with the method, and the amount of HDL-C in the serum is proportionate to the amount of Hpoprotein or apoliproprotein measured.
  • Bilirubin conjugate methodology as pertain to determining the presence of alpha lipoproteins in a sample are as follows. However, the same are not limited to the present invention, since similar techniques (e.g., using a bilirubin conjugate reagent buffered to a pH of about 5.0 - 5.2), as may be seen below, can be useful in measuring the amounts of various proteins or lipoproteins present in a serum sample.
  • Bilirubin absorption test for Alpha-Lipoprotein Bilirubin is known to bind to serum proteins and has actually been assayed using CD detection (2) , after being bound to human serum albumin (HSA) . Bilirubin is not, by itself, CD active.
  • HSA is CD active in the far UV (maxima around 218 nm) . Together in aqueous solution, the molecules form a strong association complex that absorbs and is CD active in the visible range of the spectrum. The color of the solution is not noticeably changed from that of the free bilirubin solution and the absorption spectrum of the free bilirubin and the HSA-complexed bilirubin differ only slightly. The change is too small to enable the clinical assay of either HSA or bilirubin using absorption detection. Only the complexed form has a CD spectrum and by carefully controlling the conditions, either molecule can be a reagent suitable for the assay of the other.
  • Bilirubin (and/or other organic dyestuffs) has the potential to bind to all the serum proteins.
  • HSA is the preferred host, because it is present in very large molar excess over all other proteins.
  • a detector selective enough to discriminate among the various bilirubin-protein complexes might be developed for protein recognition and for profiling serum proteins, and that full spectrum CD detection might have a significant degree of selectivity to accomplish this task.
  • preliminary evidence suggests that different CD spectra exist for bovine serum albumin . (BSA) , HSA, and gamma-globulins (GG).
  • the CD spectrum of the HSA-bilirubin complex is typically bi-modal and has a strong pH dependence, with each band reversing polarity as the pH is increased, Figure 5(a).
  • pH 5.0-5.2 roughly the center of the range around the isoelectric point for HSA, the CD spectrum is virtually baseline. Proteins with slightly different isoelectric ranges might become preferred hosts for bilirubin in the 5.0-5.2 pH range.
  • bilirubin conjugate was added to specimens of human serum and the CD spectra were measured as a function of pH.
  • the dominant spectrum at most buffered pH values was that for HSA- bilirubin complex, verified by checking it against an HSA standard (Sigma).
  • the spectrum at pH 5.0 is not typical of HSA, Figure 5(b), nor does it correspond with the spectrum for the GG-bilirubin complex.
  • a standardized procedure was developed to obtain reproducible spectra for a given serum. The details are as follows: to 200 ⁇ L of serum in a 10 L vial, add 3.0 mL of pH 5.0 buffer and 50 ⁇ L of a 1 x 10 "3 M (7mg/10mL water) solution of bilirubin conjugate (Porphyrin Products Inc. , Logan, Utah) . Shake and allow to stand for 5 minutes. Transfer the solution to a 1cm path- length, 3mL total volume, spectrophotometric cuvette and run the CD spectrum from about 575 to 375 nm.
  • the bilirubin stock solution must be prepared using distilled water and not pH 5.0 buffer, in which it rapidly oxidizes to biliverdin. The stock in water is sufficiently stable for several hours, but not overnight (w 16 hours) .
  • the alpha-lipoprotein fraction from the ISOLAB ® separator (a heparin-agarose column) , when reacted with bilirubin conjugate at a pH 5.0, was found to give a CD spectrum analogous to that for the complex with the ano ⁇ nymous "serum protein(s)".
  • the analogous spectrum was also observed for the bilirubin complex of HDL-cholesterol standard solutions obtained from Sigma Chemical Co.
  • HDL(Chug) - Cholesterol subfraction HDL-C obtained using Chugaev reagents and taking algebraic sum of CD absorption measurements at 390 and 475 nm.
  • HDL(BR) - subfraction HDL-C obtained using bilirubin conjugate at pH 5.0 and measuring directly the alpha lipoproteins associated with the HDL-C fraction, by taking CD absorption at 495 nm.
  • HDL(enz) - subfraction HDL-C obtained using the enzymatic method designated by Lab(A) and Lab(B) . * Asterisk indicates test was performed on patient's serum using mixed Chugaev reagents stored 4 weeks at
  • [ ] - brackets indicate HDL measurements which are substantially different from HDL measurements using other methods.
  • the present inventive methods have many advantageous attributes when compared with presently known methods for determining cholesterol levels, detecting steroids, etc. in test samples.
  • the present invention also encompasses novel instruments, which can allow one skilled in the art to markedly increase the speed with which the present inventive methods can be performed.
  • inventive instruments are outlined above (see Section entitled
  • one of the detection instruments encompassed hereby can simultaneously, if desired, measure HDL-C by CD absorption at a first wavelength (at about 390nm) and/or a first and a second wavelength (preferably about 390 and 475 nm) , and simultaneously if desired, measure LDL-C + VLDL-C by CD absorption at a third wavelength (preferably at about 525 nm) .
  • TC can then be determined indirectly by computer/calculator means by summation of the amounts of the cholesterol subfractions already determined.
  • Means for preparing such an instrument would include those means generally known in the art for preparing CD instruments.
  • Such an instrument may include separate detector systems for detecting CD absorbance or spectrophotometric absorption at each different wavelength monitored, if so desired.
  • absorption measurements are made at a single wavelength to determine the levels of TC and LDL-C + VLDL-C, respectively present.
  • two separate detector systems one for spectrophotometric absorbance and one for CD absorbance
  • a switching device in such an instrument which allows one to change from the CD detection mode of operation to the spectrophotometric absorption detection mode, since the two absorption measurements are taken at a single wavelength, and as such, time factors are not thought to be increased significantly by utilizing switching devices.
  • an instrument encompassed hereby can also be constructed which contains three separate detector systems, which may be used as part of a means for simultaneously monitoring the absorbance of the three different cholesterol levels in a test sample, (spectrophotometric or CD detectors system), i.e., HDL- C, VLDL-C + LDL-C (CD detector system) and TC (Spectro ⁇ photometric detector system) .
  • spectrophotometric or CD detectors system i.e., HDL- C, VLDL-C + LDL-C (CD detector system) and TC (Spectro ⁇ photometric detector system) .
  • switching device(s) can advantageously be utilized to switch between the CD mode(s) of operation and/or the spectrophotometric mode(s) of operation. The use of such switching device(s) is thought preferable in such an instrument.
  • Yet another instrument encompassed hereby can be a spectrophotometric instrument having no CD capability.
  • Such an instrument should be equipped with detectors capable of measuring the absorption of the colored products of the Chugaev reagent over a range of from about 400 - 700 nm (preferably about 450-625 nm) , or at discrete points such as at about 525 nm and 480 nm and, possibly, at about 500 nm. If automated, it should also have the capability of adding the Chugaev reagents in the order described above to reduce precipitation.
  • any such absorption spectrometer should preferably have the means to determine the levels of LDL-C + VLDL-C in a test sample by a calculation or computation from the TC and HDL-C values. It may also have the means to determine VLDL- C at about 500 nm as described above and to use that value in the computation of LDL-C in the clinical sample.
  • the TC level in a test sample could be read directly and the HDL-C level read directly after addition of an appropriate sulfate, with absorption readings being done simultaneously in two separate cuvettes after adding the basic Chugaev reagents to one tube and the basic Chugaev reagents plus sulfate additive to the second tube.
  • VLDL-C + LDL-C could then be calculated or computed automatically from the two absorption reading, if so desired.
  • spectrophotometric absorption devices such as those disclosed above, one could also determine TC, HDL- C and VLDL-C level directly.
  • test sample having the basic Chugaev reagents added thereto would be in at least a first and a second cuvettes (or tubes) and a spectrophotometric absorption reading of one of the samples taken.
  • an appropriate sulfate additive would be added to the sample in the first cuvette and an appropriate perchlorate (or dextran sulfate) added to the sample in the second cuvette, and a spectrophotometric absorption reading made of the test sample in each cuvette.
  • the level of LDL-C in the sample could then be automatically calculated from the three absorption readings, if so desired.
  • instruments herein encompassed for performing the methods of the present invention could be designed so that separate light trains exist for the CD and spectrophotometric absorption signals.
  • monochromators could be eliminated.

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Abstract

On décrit l'utilisation des méthodes spectrophotométriques, notamment l'absorption spectroscopique et le dichroïsme circulaire traditionnels, dans les procédés de détection de la chimie clinique. On décrit en particulier l'utilisation de ces méthodes spectrophotométriques pour mesurer les taux de cholestérol et mesurer directement les sous-fractions de cholestérol dans les échantillons chimiques, ainsi que pour mesurer les taux de lipoprotéines dans un échantillon d'analyse clinique et pour détecter les stéroïdes anabolisants et d'autres produits stéroïdiens. On a également prévu certains appareils de dichroïsme circulaire et de spectrophotométrie traditionnelle utiles à chacun des procédés précités.
EP19910902458 1990-01-11 1991-01-10 Circular dichroism and spectrophotometric absorption detection methods and apparatus Withdrawn EP0510064A4 (en)

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US5593894A (en) * 1990-01-11 1997-01-14 Research Corporation Technologies, Inc. Direct cholesterol assay
ATE178993T1 (de) * 1993-07-14 1999-04-15 Res Corp Technologies Inc Direkter cholesterolassay
WO1997033514A1 (fr) * 1996-03-13 1997-09-18 Hitachi, Ltd. Procede et appareil d'analyse dichromatique circulaire
EP1828781A1 (fr) * 2004-12-11 2007-09-05 Science and Technology Facilities Council Dosage pour produire un profil lipidique au moyen d'une mesure de fluorescence

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US3884638A (en) * 1973-08-01 1975-05-20 Damon Corp Method of determining cholesterol

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Title
CHEMICAL ABSTRACTS, vol. 108, no. 24, 13 June 1988, Columbus, Ohio, US; abstract no. 210070, Y. D. KHOLODOVA 'Spectral properties of steroid hormones, sapogenins and alkaloids in the Chugaev reaction as a function of their structure.' page 357 ;column 2 ; *
CHEMICAL ABSTRACTS, vol. 84, no. 19, 10 May 1976, Columbus, Ohio, US; abstract no. 132207, Y. D. KHOLODOVA ET AL. 'Chugaev's reaction for sterols.' page 211 ;column 1 ; *
CHEMICAL ABSTRACTS, vol. 98, no. 20, 16 May 1983, Columbus, Ohio, US; abstract no. 166955, Y. D. KHOLODOVA 'Chugaev's reaction for the analysis of steroids.' page 385 ;column 2 ; *
See also references of WO9110892A1 *

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CA2073681A1 (fr) 1991-07-12

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