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
  • G01N33/70—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving creatine or creatinine
• • 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
  • Y10T436/147777—Plural nitrogen in the same ring [e.g., barbituates, creatinine, etc.]

Definitions

• the deproteinization is etiected with trichloroacetic acid, the resulting supernatant is successively treated with picric acid and (aq) sodium hydroxide, and the creatinine content of the initial serum is then determined by measuring the extinction at 530-550 m
• the picric acid and the sodium hydroxide solution must be added as two successive separate pipette additions. This necessity of using the pipette twice detracts from the accu racy of the determination and requires, in the usually conducted series of tests, a substantial increase in the time and work involved in the determination. Furthermore, a relatively large quantity of serum is necessary for these conventional creatinine determinations.
• Another object is that the color reagent permit light absorption readings which can be accurately and reproducibly translated into creatinine concentrations.
• a further object is to provide a color reagent which can be premixed in advance of creatinine determinations; it should be capable of addition to the specimen in a single addition, thus avoiding the possibility of plural measuring errors and reducing the overall time required.
• a still further object is to provide a reagent which can be metered and employed in dry form.
• An additional object is to provide a process to measure creatinine concentration, particularly without diluting a specimen with water, as required by conventional methods.
• a color reagent comprising lithium picrate and lithium hydroxide is employed for the quantitative determination of creatinine by measuring light absorption of formed creatinine picrate.
• the color reagent makes it possible to determine the creatinine content of a fluid within a short period, e.g. 8-10 minutes, after adding said reagent to the fluid. Only a small sample of fluid, e.g. 0.5 milliliter (ml.) of serum, is required for an accurate creatinine determination with the reagent.
• the light absorption can be measured at any desired wavelength between 500 and 550 millimicrons (ma), especially at the favorably positioned wavelength of 546 m (measured by conventional filter photometers with a mercury lamp).
• the color reagent of this invention is particularly suitable for the determination of creatinine in biological fluids, such as serum, blood, plasma, urine and cerebrospinal fluid, as Well as for samples of foodstuffs and feed, such as meat extract, which can contain creatinine.
• biological fluids such as serum, blood, plasma, urine and cerebrospinal fluid
• samples of foodstuffs and feed such as meat extract
• synthetic creatinine required, in particular, for the preparation of standard solutions for the creatinine determination in biological fluids can be conducted readily and accurately with said color reagent.
• Biological fluids evaluated for creatinine content are first, if required, deproteinized in a conventional manner by means of a strongly acidic compound. Trichloroacetic acid and perchloric acid are especially suitable for deproteinization. Trichloroacetic acid is preferred.
• acidic compound precipitates protein; thus-precipitated protein is removed by centrifugation or another conventional separation method from biological fluids.
• the color reagent can be present in a liquid solution or in the solid phase.
• the light absorption of the creatinine picrate (formed by the reaction of said color reagent with creatinine in the fluid being analyzed) is measured in a pH range of about 9-44, preferably at a pH value of about 12. Accordingly, it is suitable to adjust the pH value of the alkaline lithium picrate solution (the color reagent) so that, after the deproteinizing agent has been neutralized, there is sufficient excess alkali present to obtain the above-mentioned favorable pH range during the color reaction.
• the weight ratio of lithium picrate to lithium hydroxide is 03 to 0.811, preferably 0.55 to 0.65:1, respectively.
• a solution of the color reagent is prepared from a picric acid solution and an excess of a lithium hydroxide solution.
• picric acid i.e. 0.5 gram (g.) per 100 g. of solution
• picric acid a saturated (at room temperature, i.e. 20 C.) picric acid solution (picric acid content approximately 1.2%) is used.
• a picric acid solution adjusted to a weakly acidic pH by the incorporation therein of lithium hydroxide is of special advantage.
• the picric acid is present, in part, as lithium picrate. Even upon cooling, for example, to temperatures of about (3., picric acid does not crystallize out of such a solution. In this manner, a stable picric acid solution of a higher concentration, e.g. 3%, can be produced.
• the lithium hydroxide solution employed advantageously contains about 8-12%, preferably about 10%, lithium hydroxide.
• the color reagent is obtained in ready-for-use form by mixing the picric acid solution, which optionally contains lithium hydroxide, with the lithium hydroxide solution.
• the picric acid solution which optionally contains lithium hydroxide
• 1.2% picric acid solution is mixed with a 10% lithium hydroxide solution.
• Said color reagent should be prepared within a short time prior to use. Most advantageously, the several solutions are mixed immediately before use. However, it is possible to store the thus-prepared mixed reagent for as long as 24 hours before use.
• the aqueous solution of the reagent can also contain an inert organic solvent miscible with water, such as ethanol.
• an organic solvent miscible with water, such as ethanol.
• the addition of such an organic solvent generally does not afford any special advantage.
• Ready-for-use solution of the components of the color reagent is alternatively prepared, for example, by adding lithium hydroxide in solid form to a solution of picric acid.
• the ready-for-use solution of the color reagent can also be freeze-dried in a conventional manner for increasing the stability thereof.
• Such a solid form of the color reagent need merely be dissolved in the required amount of water before it is used.
• a freeze-dried solid preparation of the color reagent has advantages over a mixture of solid picric acid and solid lithium hydroxide because material which might give rise to turbidity after the addition of water could be filtered 01f before freeze drying.
• the color reagent can also be prepared in solid form by premixing the solid picric acid and the solid lithium hydroxide. Due to the explosiveness of picric acid and picrates, special precautionary measures must be employed, however. It is recommended that the picric acid be mixed with inert diluents, as is customary in conjunction with explosive substances.
• Preferred diluents are water-soluble inert salts, such as water-soluble alkali salts, e.g. lithium, sodium and potassium halogenides, such as lithium chloride, bromide or iodide, sodium chloride, potassium chloride, or suitable water-soluble sulfates, e.g. sodium sulfate.
• the components can be selected so that a metered dosage of the reagent is exactly sufficient for one individual determination. It is then only necessary to add a dosage of the color reagent during each creatinine evaluation. In such a case, the absolute value would be 1 to 50 mg. preferably '20 mg. lithium picrate and 3 to mg. preferably 33 mg. lithium hydroxide.
• the color reagent is added to a fixed amount of the sample (previously deproteinized if it initially contained protein). After an incubation period of from about 5-20 minutes, preferably about 8-12 minutes, the light absorption (extinction or transmission) of the thusformed creatinine picrate is measured. Preferably, the extinction is measured advantageously in a range between 500 and 550 me, for example, at 520 m by means of a spectrophotometer, at 546 m by means of a filter photometer with a mercury lamp, or at 509 me by means of a filter photometer with a cadmium lamp.
• the sample should be maintained at about l0-30 C., preferably at about 20-25 C. during the evaluation.
• the measured light absorption is proportional to the amount of creatinine contained in the sample.
• Determinations of creatinine in deproteinized biological fluids can be unequivocally reproduced, even if Varying amounts of acid are employed for the deproteinization. For example, up to about 20% trichloroacetic acid has been employed for deproteinization without a substantial difference in the values obtained during the subsequent creatinine determination by said color reagent. Likewise, the determination of creatinine in an acid-free aqueous solution yields approximately the same results as the determination conducted in a solution previously deproteinized by an acid.
• EXAMPLE 1 One part of an aqueous 10% lithium hydroxide solution (solution I) is mixed with 5 parts of a picric acid/ lithium hydroxide solution (solution II) produced from 100 ml. of aqueous 1.2% picric acid solution and 1 ml. of solution I).
• EXAMPLE 2 One part of an aqueous 9% lithium hydroxide solution is mixed with 5 parts of an aqueous 1.2% picric acid solution.
• EXAMPLE 3 One part of a 12% aqueous lithium hydroxide solution is mixed with 5 parts of an aqueous 1.2% picric acid solution.
• EXAMPLE 4 One part of a 10% aqueous lithium hydroxide solution is separately mixed with 5 parts of a 0.5% and with 5 parts of a 1.0% aqueous picric acid solution, respectively.
• a aqueous lithium hydroxide solution is separately mixed with 5 parts of an aqueous, weakly acidic 2% and with 5 parts of an aqueous, weakly acidic 3% picric acid solution (prepared by adjusting a 2% and a 3% picric acid solution, respectively, to a weakly acidic pH, e.g. 3 to 7, by means of lithium hydroxide), respectively.
• EXAMPLE 6 100 milligrams (mg) of solid picric acid, 167 mg. of solid lithium hydroxide (obtained by evaporating a clear aqueous lithium hydroxide solution) and 100 mg. of lithium chloride are mixed. Before this mixture is utilized, it is dissolved in 10 ml. of distilled water.
• 0.5 ml. of serum is deproteinized in a conventional manner by the addition thereto of 0.5 ml. of a 10% aqueous trichloroacetic acid solution. The mixture is centrifuged for 10 minutes.
• 0.5 ml. of a standard solution containing 10 mg. of creatinine per 100 ml. of solution is likewise mixed with 0.5 ml. of a 10% aqueous trichloroacetic acid solution.
• a blank sample is produced from 0.5 ml. 10% aqueous trichloroacetic acid solution and 0.5 ml. of water.
• 0.5 ml. of the deproteinized serum sample and of the standard and blank samples, respectively, are separately mixed with 0.5 ml. of the color reagent of Example 1 and allowed to stand at 20-25 for 10 minutes. Within another 10 minutes, the extinctions of the serum sample and the standard sample are measured as compared to the blank sample (at a cuvette layer thickness of 0.5-2 cm.) by means of a spectrophotometer between 500 and 550 m preferably at 520 m or, for example, at Hg 546 or Cd 509.
• the creatinine content is calculated in accordance with the following formula:
• Analogous creatinine determinations of serum are likewise conducted with the color reagents of Examples 2 to 7, respectively.
• 1 ml. of 5% trichloroacetic acid is employed during the deproteinization in place of 0.5 ml. of 10% trichloroacetic acid, and 1 ml. of the protein-free residue (as compared to the above-employed 0.5 ml.) is added to the dry color reagent for purposes of the determination.
• EXAMPLE 10 Creatinine determination in the urine A creatinine determination is conducted analogously to that of Example 9, with a deproteinized urine sample diluted 100-fold.
• the creatinine content is calculated in accordance with the following formula:
• Creatinine content of the urine diluted to 100 times its original volume by means of water:
• a composition for the detection of creatinine which consists essentially of lithium hydroxide and at least one member selected from the group consisting of picric acid and lithium picrate, the weight ratio of said lithium hydroxide to said member being 0.3:1 to 0.8:1 respectively, wherein said ratio is calculated on the basis of said member being lithium picrate.
• a solid color reagent comprising the composition of claim 1 wherein said at least one member is picric 8 acid, and further comprising inert water-soluble solid References Cited diluent to reduce the explosive nature of the picric acid. Clark et a1" AnaL Chem 21 Na 10 October, 1949,
• a composition according to claim 1 comprising an 1218 1221 intimate admixture of components, said lithium hydroxide being present in an amount sufficient to yield a pH of 5 MORRIS WOLK, primary Examiner 4.
• U S Cl X R S. A composition as defined by claim 1 wherein said member is lithiumpicrate. 10 23--230; 260622 6.

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Cited By (5)

• 1967
  • 1967-07-20 DE DE19671648977 patent/DE1648977A1/de active Pending
• 1968
  • 1968-06-10 GB GB27507/68A patent/GB1219624A/en not_active Expired
  • 1968-06-26 NL NL6809009A patent/NL6809009A/xx unknown
  • 1968-07-04 CH CH995668A patent/CH499521A/de not_active IP Right Cessation
  • 1968-07-09 FR FR1578234D patent/FR1578234A/fr not_active Expired
  • 1968-07-10 SE SE09484/68A patent/SE334248B/xx unknown
  • 1968-07-17 US US745389A patent/US3557018A/en not_active Expired - Lifetime
  • 1968-07-17 BE BE718190D patent/BE718190A/xx unknown

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