Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
  • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase

Definitions

• This invention relates generally to a heat fractionation technique applied to serum samples to fractionate the isoenzymes which are found in the lactic acid dehydrogenase (LDH) component. More particularly, it relates to a rapid fractionation of the serum LDH by heating the serum sample for a short period of time at a pH of from 7 to in the presence of a buffer solution at a concentration which permits substantial inactivation of certain or specific isoenzyme molecules.
• LDH lactic acid dehydrogenase
• LDH is an enzyme found in human tissue, particularly abundant in kidney, cardiac, hepatic and muscular tissues. Since the LDH level remains relatively constant in normal serum samples, its elevation is indicative of tissue disfunction or disease.
• LDH activity in a blood sample relies on the use of lactate as the substrate in alkaline solution which causes the concurrent reduction of nicotinamide adenine dinucleotide (NAD) to its reduced form, i.e., NADH. This latter conversion is measured photometrically.
• NADH nicotinamide adenine dinucleotide
• the concentration of enzyme (LDH) is directly proportional to the increase in NADH concentration.
• the above sample to be assayed for LDH activity is generally heated at 37C. at a pH of 9 for a period of about 6 minutes.
• the spectrophotometric determination is then carried out.
• LDH is comprised of five isoenzymes, designated for the sake of simplicity as LDH LDH LDH LDH,, and LDH These isoenzymes have been separated and identified by electrophoresis.
• the importance in knowing of the existence of these isoenzymes is manifested in its application as a diagnostic tool. More particularly, the proportion of different isoenzyme fractions (i.e., the isoenzyme pattern) varies from tissue to tissue. For instance, cardiac extracts exhibit a pattern where fractions LDH and LDH predominate.
• hepatic tissue shows a preponderance of the LDH fraction.
• the LDH level increases in the serum even before the total LDH activity exceeds normal limits and this may facilitate early diagnosis. Furthermore, whereas total LDH activity remains elevated for an average of ten days after infarction, the LDH isoenzyme elevation commonly persists into the third week.
• Another ofthe isoenzymes, namely LDH is found to increase in activity in acute and chronic liver disorders.
• Another method practiced in the art relates to heat fractionation of LDH into the isoenzyme components whereby certain LDH isoenzyme fractions such as LDH, and LDH,; are stable at temperatures as high as 65C. whereas fraction LDH is inactivated at 57C. and LDH 3 and 4 become inactivated somewhere between 57C. and 65C. Because of this heat stability differential, one can make comparisons of the heat-stable and heat-labile LDH isoenzyme fractions. In this manner, one is permitted to assess the relative amounts of various isoenzyme fractions found within LDH.
• the heat-labile LDH, fraction (destroyed by heating at 59C.) constitutes about 10% of the total LDH activity in normal serum. Accordingly, an increase in LDH activity greater than 30% of the total (usually a reading of 3070%) provides good indication that an acute or chronic liver disorder may be present in the patient.
• the combined activity of the LDH, and LDH, fractions comprises or more of the total LDH activity, heart disfunction or disease is indicated.
• test is based on a comparative ratio between the total LDH activity and the heat labile or heat stable fractions.
• the prior art method of fractionating the LDH in the serum sample consisted of heating the undiluted serum sample at 56C, the temperature at which the LDH fraction is inactivated, for a period of about 30 minutes at a pH of about 7.0, the pH of the sample.
• the present invention is directed to the elimination of these disadvantages. If effectively permits the analyses of numerous samples on continuous stream at a rate feasible for continuous stream apparatus, and just as important, allows for an immediate determination of LDH fractionated activity with only a simple intervening heating step.
• the present invention therefore makes the heat fractionation technique commercially practicable.
• This invention relates to an effective method of fractionating LDH in a serum sample which comprises heating said sample for a period of from about 0.5 to about 5 minutes at a temperature of from about 55C to about 65C at a pH in the range from 7 to l0 maintained by an aqueous buffer solution, said buffer concentration having an ionic strength sufficient to substantially inactivate the isoenzyme molecules and subsequently quick-cooling the resulting mixture.
• a buffer consisting of a 0.67 M aqueous solution of aminomethyl propanol
• the unique feature of the herein disclosed method of heat fractionating LDH in a serum sample as a means for diagnosing certain body disorders is its ability to be adapted to a continuous stream process whereby numerous samples are assayed consecutively. After rapid on-line heat fractionation and cooling, the sample is in condition for residual LDH assay.
• the end result is derived from a comparison between the total LDH assay and the LDH fractionation assay. Therefore, if these steps are carried out consecutively or simultaneously on parallel dual channels without an undesirable waiting period, the overall process is not only a time saver, it also lends itself to a continuous stream analyzer.
• the present process provides the integration of these steps. This integration is brought about by the rapid heating step (about 40 seconds) and subsequent quickcooling step prior to LDH fractionation assay.
• the total and fractionated LDH activity can be measured under the same pH and buffer conditions.
• An aliquot of serum is assayed for total LDH at a pH of 7 to 10 and about 37C.
• An identical aliquot is rapidly heated in the same range from 55C to about 65C, preferably about 625C, cooled and assayed for LDH residual activity.
• the heating step may require a longer time period. It is possible that under certain conditions, the heating step may take as long as 10 or 15 minutes. Of course, it must be recognized that beyond about minutes the heating step will be of very little value if a continuous step process is to be used.
• EXAMPLE I EXAMPLE ll
• the procedure of Example I is repeated on a serum sample from a patient with mycardial infarct. Electrophoretic analysis showed elevated LDH l and LDH 2 isoenzymes. The fractionated LDH values according to the hereindisclosed procedure was greater than 85%.
• Example Ill The procedure of Example I is repeated. On a serum sample taken from a patient having liver disease. Electrophoretic analysis showed elevated LDH 4 and 5. Analysis by the herein described procedure showed a fractionated LDH value of 50%.
• Example IV The procedure of Example l is substantially repeated using the following buffers in lieu of aminomethyl propanol:
• a method for effectively determining LDH activity in a serum sample so as to diagnostically determine the existence and presence of diseased organs comprising: mixing the serum sample with an aqueous buffer solution heating said serum sample-buffer solution mixture for a period of from 0.5 to about 5 minutes at a pH in the range from 7.6 to at a temperature sufficient to fractionate the LDH, said aqueous buffer solution having an ionic strength sufficient to substantially inactivate particular isoenzyme molecules during heating, rapidly cooling the serum sample-buffer solution to a temperature at least below 37C, and assaying said cooled sample for residual LDH activity at substantially said pH.
• said buffer solution is a borate-containing aqueous solution.
• a method of effectively determining LDH activity in a serum sample to diagnostically determine the existence and presence of diseased organs which comprises heating said sample for a period of from 0.5 to about 5 minutes at a temperature of from about 55C. to about C. at a pH in the range from 7.6 to H) maintained by an aqueous buffer solution, said buffer concentration having an ionic strength sufficient to substantially inactivate the isoenzyme molecules, followed by cooling of the resulting mixture to a temperature at least below 37C., and assaying said cooled sample for residual LDH activity at substantially said given 15.
• the method according to claim 14 wherein said fractionation is effected within a period of about 40 seconds.

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• 1972
  • 1972-06-09 US US261500A patent/US3862010A/en not_active Expired - Lifetime
• 1973
  • 1973-04-26 NL NL7305801A patent/NL7305801A/xx unknown
  • 1973-04-27 CA CA169,753A patent/CA999507A/en not_active Expired
  • 1973-05-04 BE BE130766A patent/BE799119A/xx unknown
  • 1973-05-14 AU AU55636/73A patent/AU5563673A/en not_active Expired
  • 1973-06-01 DE DE2327893A patent/DE2327893A1/de active Pending
  • 1973-06-06 JP JP48063093A patent/JPS4961382A/ja active Pending
  • 1973-06-07 FR FR7320682A patent/FR2187803B3/fr not_active Expired
  • 1973-06-11 IT IT1011/73A patent/IT992068B/it active

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