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
  • G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
  • G01N33/728—Bilirubin; including biliverdin
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/903—Diazo reactions
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S534/00—Organic compounds -- part of the class 532-570 series
  • Y10S534/03—Polymeric azo compounds or azo compounds containing polymeric moieties
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T436/00—Chemistry: analytical and immunological testing
  • Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
  • Y10T436/145555—Hetero-N
  • Y10T436/146666—Bile pigment

Definitions

• ABSTRACT A method for the spectrophotometric determination of bilirubin in fluids, particularly body fluids, such as blood serum.
• the process comprises reacting the bilirubin-containing fluid with a mixture of diazosulfanilic acid-l,5-napht:halene disulfonate and ethylene or propylene glycol :in aqueous mineral acid to form a chromophore, and thereafter measuring the absorbance of the chromophore after the reaction is complete by means of a centrifugal analytical photometer.
• concentration in an unknown sample can be conveniently calculated by comparison with the results obtained from the simultaneous measurement of a sample of known concentration.
• This invention relates in general to a process for the determination of bilirubin in fluids, particularly body fluids.
• the invention relates to a process for the determination of bilirubin in blood serum.
• the invention relates to a process for the determination of bilirubin using a centifugal analytical photometer.
• bilirubin in body fluids such as blood serum
• body fluids such as blood serum
• the determination of bilirubin in serum is important in several diseases, in particular hepatitis and erythroblastosis fetalis, where bilirubin elevation occurs.
• bilirubin in biological samples is still determined photometrically employing variations ofa single basic procedure involving the use of diazotized sulfanilic acid which reacts with bilirubin to form a chromophore (Evelyn, K.A. and Malloy, H.T. J.
• a second widely used variation of the procedure employing diazotized sulfanilic acid as a test for bilirubin involves the use of caffeine as an accelerator. While the need for serum dilution and blank measurements is eliminated by this technique, the use of caffeine requires that the reaction mixture be made alkaline in order to shift the absorbance of the resulting chromophore to a longer wavelength so that it can be determined photometrically. As a result, this process, as in 2 the case when methanol is used as an accelerator, is slow, tedious, complicated and incompatible with clinical testing techniques.
• Another object of this invention is to provide a process wherein many of the disadvantages indicated above are eliminated or minimized.
• Another object of this invention is to provide a process for the determination of bilirubin which is much more rapid than those currently in use.
• a further object of the invention is to provide a process for the determination of bilirubin which utilizes a centrifugal analytical photometer.
• a still further object of the invention is to provide a process for the determination of bilirubin which employs a stable diagnostic reagent which can be packaged in a convenient ready-to-use form.
• An additional object of the invention is to provide a reagent which when added to a bilirubin-containing serum generates a chromophore whose absorbance is linearily related to bilirubin concentration, and whose use does not require dilution and a blank absorbance measurement on the test sample prior to use.
• Yet another object of the invention is to provide a process which does not require a blank absorbance measurement on the diagnostic reagent employed, or that the reaction mixture be made alkaline or treated with another reagent, in order to permit determination of bilirubin photometrically.
• the purpose of the ethylene or propylene glycol is that of an accelerator for the reaction between the diazosulfanilic acid-1,5- naphthalene disulfonate and bilirubin, but does not introduce any of the disadvantages associated with other accelerators such as methanol and caffeine.
• the diazosulfanilic acid-1,5-naphthalene disulfonate is stable over long periods of time and can be stored in closed vials for at least six months at room temperature. As a result, this material can be packaged and sold as a convenient ready-for-use" diagnostic reagent.
• the invention is directed to a process for the determination of bilirubin in fluids.
• the process comprises the steps of:
• the absorbance of the purple colored complex produced by the reaction of bilirubin and diazosulfanilie acid-1,5-naphthalene disulfonate in an aqueous mineral acid solution can be measured in accordance with the instant invention by means of a centrifugal analytical photometer, and the amount of bilirubin present can be quantitatively determined by a comparison of this absorbance with that of a complex prepared in like manner from a fluid containing a known concentration of bilirubin.
• Analytical photometers which utilize a centrifugal field have recently become available for the rapid microanalysis ofa wide variety of liquids, such as body fluids, e.g., blood serum, food products, and the like.
• Reagents are placed in the innermost cavity and serum samples in the center cavity of the sample loading disk which is then indexed and positioned in the rotor with each reagent and serum sample having its respective cuvette.
• centrifugal force moves the reagents and sample to the outermost cavity where they are transferred through a small channel to the curvette.
• the reagent and sample mix.
• the filled cuvettes rapidly spin past the fixed light beam and the transmission of light is measured.
• a centrifugal rotary photometer supplied by Union Carbide Corporation under the trademark CentrifiChem was utilized.
• a Teflon disk containing samples and reagents is inserted into a rotor with 30 radially arranged cuvettes.
• the reagent rises from every individual reagent well up to individual sample cavities, and the sample reagent mixture is transferred into the single cuvettes within 1.5 seconds.
• One cuvette containing water is used as a reference when the cuvettes spin past the stationary light beam of a spectrophotometer which measures the absorbance and displays it on an oscilloscope.
• Two sets of digitized absorbance readings of each cuvette can be stored simultaneously and the difference between them processed in a computer.
• a reading is taken of the first absorbance after start and the time interval after which the second measurement occurs also noted.
• a number of readings can be taken after the initial one.
• the absorbance change per interval is expressed in AA/min.
• the absorbance differences between initial and every individual subsequent set of readings is measured.
• This approach allows automatic compensation for cuvette to cuvette variations and for serum and reagent blanks if readings are taken before the reaction has started to a measurable degree.
• a blank reading recorded in a preceding run can be used as initial reading in a subsequent run. After a selected time interval has elasped, the readings are printed out.
• the last set of data stored in the memory can be multiplied by means of the computer which permits direct print-out in concentration units.
• the rotor is thermostated within i O.lC. in an air bath.
• sample having a bilirubin concentration within the range up to 20 milligrams per hundred milliliters with 50 microliters of diluent and 250 microliters of the diagnostic reagent solution.
• the amount of sample and reagent can be varied depending upon the concentration of reagent, amount of bilirubin present and the particular analyzer employed. For example, ratios of microliters of sample to microliters of diagnostic reagent solution of from 1:5 to 1:25 can be employed.
• the absorbance is measured after reaction between the bilirubin and diazosulfanilic acid-1,5-naphthalene disulfonate is complete, and compared to the absorbance obtained simultaneously from a mixture containing a known concentration of bilirubin.
• the accelerator employed ethylene glycol or propylene glycol
• serum/ water a known concentration of bilirubin
• the mixture of diazosulfanilic acid-1,5-naphthalene disulfonate and ethylene or propylene glycol in aqueous mineral acid employed as diagnostic reagent for bilirubin according to the present invention can be conveniently prepared by dissolving from 125 milligrams to 400 milligrams of diazosulfanilic acid-l,5-naphthalene disulfonate in mixtures containing from 3 milliliters to 25 milliliters of 0.5 N to 1.0 N HCl and 75 milliliters of ethylene or propylene glycol.
• a suitable reagent can be produced by dissolving 150 milligrams of diazosulfanilic acid-1,5-naphthalene disulfonate in a mixture of 75 milliliters of ethylene glycol and 3.6 milliliters of 1.0 N hydrochloric acid.
• the diazosulfanilic acid-1,5-naphthalene disulfonate employed in the diagnostic reagent was prepared as follows: l.73 grams (0.01 moles) of p-sulfanilic acid were added to a well stirred mixture of 3 milliliters of distilled water, 0.1 grams of a dispersing agent (Ultravon) and 3 grams of an aqueous solution containing 33-38% hydrogen chloride. After a good suspension was obtained, five grams of ice were added and the mixture was placed in an ice bath until the temperature reached 5C. One and seven-tenth milliliters (1.7 ml.) of a 40 percent by weight aqueous solution of NaNo were then rapidly added under the surface of the suspension. The temperature of the suspension was maintained at 5-7C. for minutes, and then at room temperature for another 15 minutes. The addition of the NaNo solution caused most of the solid present to dissolve, and
• a standard bilirubin reference solution can be prepared by dissolving milligrams of bilirubin in 100 milliliters of 0.1 N Na CO
• the following example is illustrative:
• EXAMPLE l A blank value was determined for each cuvette of a CentrifiChem Automatic Analyzer and stored in the analyzer until the actual test was performed. These values were obtained by loading the reagent cavities of the transfer disc sample holder with 400 microliters of water in each position, spinning it into the cuvettes, and allowing the instrument to record the absorbance of each cuvette after three seconds on a data channel.
• Sample cavities l and 2 of the transfer disc sample holder of the CentrifiChem Automatic Analyzer were then loaded with 25 microliters of a bilirubin standard (containing 20.3 milligrams of billirubin per 100 milliliters of 0.1 N Na; CO and each sample was diluted with an additional 50 microliters of distilled water.
• the remaining sample cavities, i.e., cavities 3-29, were each loaded with 25 microliter samples of human sera diluted with 50 microliters of distilled water.
• Each reagent cavity of the sample holder i.e., l-29 was loaded with 250 microliters of a mixture containing 25 milligrams of diazosulfanilic acid-l ,5, -naphthalene disulfonate per l3.l milliliters of a mixtuer of ethylene glycol and 1.0 N HCl in a 20:1 volume ratio.
• the reference 6 position contained 400 milliliters of distilled water in the innermost cavity.
• the loaded disc was placed in the rotor and covered. Because of differences in viscosity between ethylene glycol and serum/water, the solutions were given an extra mixing by simultaneously pressing the spin" and mix buttons for 4 seconds (pressing the mix button draws a stream of bubbles through each cuvette to allow proper mixing of the contents thereof).
• the instrument read the absorbance of each cuvette, and automatically substracted from it the corresponding stored blank value. Absorbance was measured with a 550 nm interference filter.
• the absorbance of the samples compared to the standard was used to determine bilirubin concentrations. According to Beers Law, the absorbance of a solution is proportional to the concentration of the solutions chromophore as long as the concentration is low. Accordingly, the concentration. of bilirubin complex formed in the reaction is at any time proportional to the absorbance it generates. in turn, the concentration of the bilirubin complex is proportional to the original concentratiton of bilirubin.
• the absorbance In order to convert absorbalnce into concentration the absorbance has to be multiplied with a factor F (Absorbance X F Concentration). In the analyzer employed this is achieved by setting the appropriate factor on a digital switch which automatically multiples the absorbance whereupon concentration units will be printed out directlty.
• F Absorbance X F Concentration
• a process for the determination of bilirubin in a bilirubin-containing fluid which comprises the steps of:
• eral acid is hydrochloric acid.

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Cited By (4)

Citations (2)

• 1972
  • 1972-12-14 US US314977A patent/US3923459A/en not_active Expired - Lifetime
• 1973
  • 1973-10-25 CA CA184,206A patent/CA1020550A/en not_active Expired

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