US3862010A - Method of heat fractionating ldh into isoenzyme components - Google Patents

Method of heat fractionating ldh into isoenzyme components Download PDF

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US3862010A
US3862010A US261500A US26150072A US3862010A US 3862010 A US3862010 A US 3862010A US 261500 A US261500 A US 261500A US 26150072 A US26150072 A US 26150072A US 3862010 A US3862010 A US 3862010A
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ldh
buffer solution
aqueous
buffer
solution
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US261500A
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Robert L Rush
Anne C Delea
Milos Stastny
Edward D Lash
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Bayer Corp
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Technicon Instruments Corp
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Priority to NL7305801A priority patent/NL7305801A/xx
Priority to CA169,753A priority patent/CA999507A/en
Priority to BE130766A priority patent/BE799119A/en
Priority to AU55636/73A priority patent/AU5563673A/en
Priority to DE2327893A priority patent/DE2327893A1/en
Priority to JP48063093A priority patent/JPS4961382A/ja
Priority to FR7320682A priority patent/FR2187803B3/fr
Priority to IT1011/73A priority patent/IT992068B/en
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    • 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.

Abstract

A method of effectively heat fractionating lactic acid dehydrogenase-containing samples into their isoenzyme components to determine abnormal LDH activity for diagnostic purposes is disclosed which relates to a rapid heating step in the presence of a buffered solution having a certain concentration and carried out at a specified pH range.

Description

United States Patent [1 1 Rush et al.
[ 1 Jan. 21, 1975 METHOD OF HEAT FRACTIONATING LDH INTO ISOENZYME COMPONENTS [75] Inventors: Robert L. Rush, Spring Valley; Anne C. Delea, Yonkers; Milos Stastny, Ossining; Edward D. Lash, Tarrytown, all of N.Y.
[73] Assignee: Technicon Instruments Corporation,
Tarrytown, N.Y.
[22] Filed: June 9, 1972 [21] Appl. No.: 261,500
[52] US. Cl. 195/1035 R, 195/66 R [51] Int. Cl G01n 31/14, GOln 33/16 [58] Field of Search 195/1035 R [56] References Cited OTHER PUBLICATIONS Strandjord et al., J. Am. Med. Ass0cn.,
182(1l):l099-l102, (1962).
Don et al., Enzymologia," 36(6):353-370, (1969).
Vesell et al., Proc. Nat. Acad. Sci, 56zl3l7-l324, (1966).
Primary ExaminerAlvin E. Tanenholtz Attorney, Agent, or FirmMorgan, Finnegan, Durham & Pine 1 ABSTRACT 24 Claims, N0 Drawings METHOD OF HEAT FRACTIONATING LDH INTO ISOENZYME COMPONENTS BACKGROUND OF THE INVENTION 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 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.
The determination of 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. The concentration of enzyme (LDH) is directly proportional to the increase in NADH concentration.
Procedurally, 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.
Recently, it was discovered that 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.
Correspondingly, hepatic tissue shows a preponderance of the LDH fraction.
The usefulness of these observations can be applied practically since an increase in normal serum LDH levels is ascribable to an increase in one or more isoenzyme fractions which in turn may be traced to the affected tissue.
For example, after myocardial infarction, 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.
The most usual procedure for separation of the LDH isoenzymes is electrophoresis and is still regarded as the standard in such measurements. However, for practical use, it is unacceptable because of the time and labor involved.
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.
If the combined activity of the LDH, and LDH, fractions (heat-stable) comprises or more of the total LDH activity, heart disfunction or disease is indicated.
It is apparent from the foregoing discussion that the 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 obvious drawback of the procedure in highly so phisticated continuous stream diagnostic instruments is apparent. Where hundreds of samples are to be tested, it would be impractical to heat each one for 30 minutes after assaying for total LDH activity. Such restriction would preclude application to a continuous stream apparatus.
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.
SUMMARY OF THE INVENTION 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.
Preferred embodiments of the above-described process comprise:
a. A heating period of about 40 seconds;
b. A buffer consisting of a 0.67 M aqueous solution of aminomethyl propanol;
c. A mixture pH of about 9;
d. A mixing temperature of about 625C; and
e. A cooling period of 30 to 60 seconds.
DETAILED DESCRIPTION OF THE INVENTION 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.
It turns out there are four critical variables in the herein disclosed process, all interrelated with one another. Accordingly, the optimum conditions for a fixed pH in the range of 7-10 must necessarily be empirically determined.
It is important to realize that the pH during fractionation and ensuing assay be the same as that of the total LDH assay.
For example, it is found that when the pH is set or fixed at 9, which is preferred, a heating period of about 40 seconds at a temperature of approximately 6263C is mandated when a buffer comprising aminomethyl propanol is used. Moreover, a 0.67 M solution is necessary to provide a concentration of buffer having an ionic strength sufficient to substantially inactivate certain or specific isoenzyme molecules.
If the pH is less than 9, 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.
correspondingly, if the pH is above 9 the heating time will be shortened. However beyond a pH of 10, the fractionation proceeds so rapidly as to be impractical. Moreover, adverse chemical reactions take place which result in misleading assays.
Since the mixture during heating must be restricted to a pH in the range of 7-10, and preferably 9, numerous buffer compositions become readily apparent to one skilled in the art.
For instance, the following list, by no means exhaustive, enumerates representative buffers suitable for inclusion into the subject process:
tris (hydroxymethyl) aminomethane glycine triethanolamine borate-containing buffer ammonium-containing buffer Each of these buffer compositions is capable of maintaining a pH value in the range of 7-10. Once the pH is set, the other variables can then be determined to provide the optimum conditions for that buffer and pH level. This determination is not complicated and the ultimately arrived at conditions will fall within the boundaries circumscribed herein.
Accordingly, one skilled in the art can choose beforehand each of the variables at random provided they fall within the confines of the invention as described. However, to determine the optimum set of conditions. sim' 5 ple experimentation is necessary.
In sum, the unique advantages offered by the process of this invention include:
1. suitable application to a continuous stream analyzer; and 2. elimination of a costly and time-consuming prior art procedure previously in use.
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%.
These results indicate an agreement between the process of this invention and the standard electrophoretic method.
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%.
These results indicated an agreement between the process of this invention and the standard electrophoretic method.
EXAMPLE IV The procedure of Example l is substantially repeated using the following buffers in lieu of aminomethyl propanol:
tris (hydroxymethyl) aminomethane glycine triethanolamine borate-containing buffer quaternary ammonium salt-containing buffer EXAMPLE V The procedure of Example I is substantially repeated using the following heating time periods with comparable results:
20 seconds 60 seconds 3 minutes EXAMPLE V1 The procedure of Example I is substantially repeated at the following pH levels with comparable results:
What is claimed is:
l. 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.
2. The method according to claim 1 wherein prior to said heating step, an identical aliquot of the sample is colorimetrically assayed for total LDH activity.
3. The method according to claim 1 wherein said heating step is carried out at a temperature between about 55C. and 65C.
4. The method according to claim 3 wherein said temperature is about 625C.
5. The method according to claim 1 wherein said heating period is 40 seconds.
6. The method according to claim 1 wherein said pH is 9.
7. The method according to claim 1 wherein said buffer is aminomethyl propanol in the form of an 0.67 M aqueous solution.
8. The method according to claim 1 wherein said buffer solution is aqueous tris (hydroxymethyl) aminomethane.
9. The method according to claim 1 wherein said buffer solution is aqueous glycine.
10. The method according to claim 1 wherein said buffer solution is aqueous triethanolamine.
11. The method according to claim 1 wherein said buffer solution is a borate-containing aqueous solution.
12. The method according to claim I wherein said buffer solution is a quarternary ammonium saltcontaining aqueous solution.
13. The method according to claim 1 wherein said cooling is accomplished over a period of from 15 to 90 seconds.
14. 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.
16. The method according to claim 14 wherein said pH during fractionation is about pH 9.
17. The method according to claim 14 wherein said buffer is aminomethyl propanol in the form of an 0.67M aqueous solution.
18. The method according to claim 14 wherein said fractionation temperature is about 625C.
19. The method according to claim 14 wherein said buffered solution is aqueous tris (hydroxymethyl) aminomethane.
20. The method according to claim 14 wherein said buffer solution is aqueous glycine.
21. The method according to claim 14 wherein said buffer solution is aqueous triethanolamine.
22. The method according to claim 14 wherein said buffer solution is a borate-containing aqueous solution.
23. The method according to claim 14 wherein said buffer solution is a quaternary ammonium salt containing aqueous solution.
24. The method according to claim 14 wherein said cooling is accomplished over a period of from 15 to

Claims (23)

  1. 2. The method according to claim 1 wherein prior to said heating step, an identical aliquot of the sample is colorimetrically assayed for total LDH activity.
  2. 3. The method according to claim 1 wherein said heating step is carried out at a temperature between about 55*C. and 65*C.
  3. 4. The method according to claim 3 wherein said temperature is about 62.5*C.
  4. 5. The method according to claim 1 wherein said heating period is 40 seconds.
  5. 6. The method according to claim 1 wherein said pH is 9.
  6. 7. The method according to claim 1 wherein said buffer is aminomethyl propanol in the form of an 0.67 M aqueous solution.
  7. 8. The method according to claim 1 wherein said buffer solution is aqueous tris (hydroxymethyl) aminomethane.
  8. 9. The method according to claim 1 wherein said buffer solution is aqueous glycine.
  9. 10. The method according to claim 1 wherein said buffer solution is aqueous triethanolamine.
  10. 11. The method according to claim 1 wherein said buffer solution is a borate-containing aqueous solution.
  11. 12. The method according to claim 1 wherein said buffer solution is a quarternary ammonIum salt-containing aqueous solution.
  12. 13. The method according to claim 1 wherein said cooling is accomplished over a period of from 15 to 90 seconds.
  13. 14. 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 55*C. to about 65*C. at a pH in the range from 7.6 to 10 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 37*C., and assaying said cooled sample for residual LDH activity at substantially said given pH.
  14. 15. The method according to claim 14 wherein said fractionation is effected within a period of about 40 seconds.
  15. 16. The method according to claim 14 wherein said pH during fractionation is about pH 9.
  16. 17. The method according to claim 14 wherein said buffer is aminomethyl propanol in the form of an 0.67M aqueous solution.
  17. 18. The method according to claim 14 wherein said fractionation temperature is about 62.5*C.
  18. 19. The method according to claim 14 wherein said buffered solution is aqueous tris (hydroxymethyl) aminomethane.
  19. 20. The method according to claim 14 wherein said buffer solution is aqueous glycine.
  20. 21. The method according to claim 14 wherein said buffer solution is aqueous triethanolamine.
  21. 22. The method according to claim 14 wherein said buffer solution is a borate-containing aqueous solution.
  22. 23. The method according to claim 14 wherein said buffer solution is a quaternary ammonium salt containing aqueous solution.
  23. 24. The method according to claim 14 wherein said cooling is accomplished over a period of from 15 to 90 seconds.
US261500A 1972-06-09 1972-06-09 Method of heat fractionating ldh into isoenzyme components Expired - Lifetime US3862010A (en)

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US261500A US3862010A (en) 1972-06-09 1972-06-09 Method of heat fractionating ldh into isoenzyme components
NL7305801A NL7305801A (en) 1972-06-09 1973-04-26
CA169,753A CA999507A (en) 1972-06-09 1973-04-27 Method of heat fractionating ldh into isoenzyme components
BE130766A BE799119A (en) 1972-06-09 1973-05-04 HOT FRACTIONATION OF LACTICODESHYDROGENASE INTO ITS CONSTITUTIVE ISO-ENZYMES
AU55636/73A AU5563673A (en) 1972-06-09 1973-05-14 Method of heat fractionating ldh into isoenzyme components
DE2327893A DE2327893A1 (en) 1972-06-09 1973-06-01 PROCESS FOR THE HEAT FRACTION OF LACTATE DEHYDROGENASE INTO ISOENZYME COMPONENTS
JP48063093A JPS4961382A (en) 1972-06-09 1973-06-06
FR7320682A FR2187803B3 (en) 1972-06-09 1973-06-07
IT1011/73A IT992068B (en) 1972-06-09 1973-06-11 PROCEDURE FOR HOT FRACTIONING LDH INTO ISOENZYME COMPONENTS

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4003795A (en) * 1974-09-12 1977-01-18 Kommanditgesellschaft Schwarzhaupt Process for the determination of at least one of the isoenzymes of lactatendehydrogenase
US4311791A (en) * 1978-06-05 1982-01-19 Bernstein Larry H Automated kinetic determination of lactate dehydrogenase isoenzymes in serum
US4543327A (en) * 1980-06-10 1985-09-24 Bernstein Larry H Malate dehydrogenase method

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5974998A (en) * 1982-10-22 1984-04-27 Yatoron:Kk Individual method for measuring isoenzyme activity of lactate dehydroganse in human blood serum

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Oon et al., "Enzymologia," 36(6):353-370, (1969). *
Strandjord et al., "J. Am. Med. Assocn.," 182(11):1099-1102, (1962). *
Vesell et al., "Proc. Nat. Acad. Sci.," 56:1317-1324, (1966). *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4003795A (en) * 1974-09-12 1977-01-18 Kommanditgesellschaft Schwarzhaupt Process for the determination of at least one of the isoenzymes of lactatendehydrogenase
US4311791A (en) * 1978-06-05 1982-01-19 Bernstein Larry H Automated kinetic determination of lactate dehydrogenase isoenzymes in serum
US4543327A (en) * 1980-06-10 1985-09-24 Bernstein Larry H Malate dehydrogenase method

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Publication number Publication date
IT992068B (en) 1975-09-10
BE799119A (en) 1973-11-05
FR2187803B3 (en) 1976-05-28
AU5563673A (en) 1974-11-14
DE2327893A1 (en) 1973-12-13
CA999507A (en) 1976-11-09
NL7305801A (en) 1973-12-11
JPS4961382A (en) 1974-06-14
FR2187803A1 (en) 1974-01-18

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