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
  • G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors
• • 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
  • Y10T436/20—Oxygen containing
  • Y10T436/200833—Carbonyl, ether, aldehyde or ketone containing
  • Y10T436/201666—Carboxylic acid
• • 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
  • Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
  • Y10T436/25375—Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
  • Y10T436/255—Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.] including use of a solid sorbent, semipermeable membrane, or liquid extraction

Definitions

• results of the diagnostic procedure described permits from the analysis of one urine specimen the accurate and definitive identification of the presence and severity of a folic acid and/or vitamin B deficiency state and thus affords a basis for determining specifically those two deficiency states.
• Other aspects of the invention are described in the specification.
• This invention relates to a method and compositions for differential diagnosis of the megaloblastic anemia syndromes. More particularly, it pertains to the specific posi tive differential diagnosis of cobalamin (vitamin B deficiency disease syndromes from folic acid deficiency disease syndromes. Both are characterized by causing histologically and usually clinically identical megaloblastic macrocytic anemia syndromes.
• cobalamin as used herein is directed generically to the entire group or organocobalt compounds possessing votamin B activity and includes the cyano and hydroxycob-alarnins as Well as other naturally occurring related compounds, and complexes thereof, usually with proteins.
• folic acid as used herein is directed generically to the entire group of folate derivatives or pteroylglutamate compounds, including the mono, di, tri, hepta and other polyglutamates of pteroylglutamic acid, reduced forms, of each of these compounds as well as derivatives such as the formyl, methyl, methylene, methenyl and 'ice formimino, and natural complexes thereof, usually with proteins.
• folic acid will clear the megaloblastic anemia of pernicious (Addisonian) anemia caused by a cobalamin (vitamin B deficiency, but it does not affect or arrest the neurological complications which are part of the vitamin B deficiency un derlying the Addisonian anemia syndrome.
• Folic acid in amounts sufiicient to restore normal blood values in Addisonian pernicious anemias, does not arrest the progression of existing neurological complications due to vitamin B deficiency, and will not prevent the development of neurological lesions in patients that were heretofore free of them.
• folic acid With continued administration of folic acid to an unrecognized pernicious anemia patient, in addition to or besides the appearance and/or progression of neurological disease, the macrocytic megaloblastic anemia, thrombocytopenia and resulting purpura or hemorrhage, and neutropenia and infection may occur or recur.
• vitamin B may produce a temporary improvement of the anemia, if the vitamin B is in sufliciently high dosage, and the folic acid deficiency is not too severe, while permitting the folic acid deficiency to continue to the point where severe anemia or other manifestations of the folic acid deficiency state, such as thrombocytopenia and purpura or neutropenia may appear with resulting serious consequences for the patient.
• This invention provides a method for the differential diagnosis of folic acid and vitamin B deficency in humans which comprises administrating loading dosages of histidine or its non-toxic salts in combination with a precursor of methylmalonate.
• the loadng dosages are the amounts of each of these substances which will cause significant increases in the amounts of FIGLU and MMa excreted by the tissues.
• FIGLU is excreted in the urine in diagnostically increased amounts, in folic acid deficient subjects;
• MMa is excreted in the urine in diagnostically increased amounts in vitamn B deficent subjects.
• the MMa precursors which are diagnostically effective when administered in loading dosage combination with histidine are L-valne, DL-valne, L-isoleucine, DL-isoleucine, L-threonine, DL-threonine, thymine, L-homoserine, DL-homoserine, and their nontoxic complexes and salts.
• the threonine, thymine and homoserine forms are novel, in that they have never before been reported alone or in combination, as MMa precursors useful for the diagnosis of the vitamin B deficiency state in man.
• the best urinary output of significant amounts of FIGLU and MMa results when the loading dosages are administered in divided amounts within the loading period, of about 12 hours.
• the loading dosages should be administered in three portions at 4 hour intervals.
• a total loading dose of L-histidine HCl'H O should aggregate from about to 20 gm. depending upon the individuals size and weight.
• the loading dosage amount of DL-valine for example, should aggregate from about to about 60 gm.
• these combined aggregates should preferably be administered, in 3 portions with a portion given every 4 hours. The individual portions, under such an administration procedure, would range from 1.7 to 6.67 gm. of L-histidine, HCI-H O and 5 to gm. of DL-valine.
• this invention also includes compositions for the simultaneous administration of these materials in specified dosages and proportions.
• compositional aspects of this invention includes the discovery that while the L-form of the precursor compounds are active, the D-form is not active.
• an increase in MMa excretion has been found that is from 30 to greater than would be expected from the L-form content of administered loading dosage.
• Loading dosage encompasses the aggregate amounts of histidine and MMa precursors that are concurrently or simultaneously administered to cause the characteristic elevated excretion of either FIGLU or MMa in folate or vitamin B deficient individuals.
• the aggregate loading dosage of histidine, for adults, in my composition is from about 5 to about 20 gm.; and for the MMa precursor, the aggregate loading dosage is from about 10 to about gm.
• the range of the aggregate dosage amounts for the specific MMa precursors in my combination for administration to adult subjects is listed below:
• Range of aggregate loading dosage of MMa Precursors used with histidine, for adults Precursor: Grams DL-valine 15-60 DL-threonine 3 0-80 L-valine 10-40 L-threonine 20-60 DL-isoleucine 15-60 L-isoleucine 10-30 'Ihymine 1 0-50 DL-homoserine 4010() All of the above dosages ranges provided diagnostically significant increases of urinary MMa in vitamin B deficient subjects, but not in normal control subjects, folic acid deficient or other non-vitamin B deficient patients with various types of anemia.
• threonine Of great interest with relation to intermediary metabolism leading to MMa, in the human, is the increase produced by threonine, thymine and homoserine.
• the urinary MMa enhancing effect of the latter compounds has not been previously shown in humans with vitamin B deficiency.
• thymine produces urinary MMa increases of about /2, L-threonine about A to /5 that of L-valine, and DL-hornoserine about /6 to 4 that of L-valine.
• the relative order of the urinary MMa enhancing activity of the MMa substances studied, in human subjects having vitamin B deficiency, but not in folic acid deficient or control subjects, on an equimolar L-form basis are: (1) L-valine, (2) L-isoleucine, (3) DL-valine, (4) DL-isoleucine, (5) thymine, (6) L-threonine, (7) DL- threonine, (8) L-homoserine, and (9) DL-homoserine.
• L-valine and L-isoleucine When administered in equimolar amounts, L-valine and L-isoleucine produce a urinary MMa increase of about the same order of magnitude, although L-valine usually, gives moderately higher values.
• the racemic form of these amino acids when given in a molar amount equal to that of the L-form amino acid, gives urinary MMa increases in the order of 3080% that of the L-form.
• the compounds necessary for administration according to the method of this invention are generally not too palatable in the forms that are commonly available. Many of them, including histidine which is acidic to some and salty to others; valine which is not too soluble and has a slightly fatty fiavor and isoleucine which causes a lumpy feeling in the stomach actually cannot be administered suspended or dissolved in water without complaints from the patients. Many vehicles for their administration in loading dosage have been tried. I have discovered, however, an inexpensive vehicle, apple juice, which best solubilizes histidine and most of the MMa precursors, overcomes in an unusual way thepalatability and administration problems, with the FIGLU and MMa precursors.
• the loading dosages in divided portions, can easily be administered dissolved or suspended to 2 to 8 ozs. of apple juice.
• the total loading dosage according to this invention may be administered admixed in up to a quart of this vehicle.
• the palatability of the apple juice permits the administration of the loading dosages even to children and infants.
• loading dosages for infants and children where prompt diagnosis should be made due to the poor enzymatic reserves, I have found that loading doses are slightly greater than in adults when calculated on the basis of body weight.
• the loading dosage for infants and children aggregates from about 0.08 to about 0.14 gm. per pound of body weight for histidine and its related FIGLU precursors and from about 0.08 to 1.40 gm. per poundof body weight for the MMa precursors.
• the specific dosage for the latter is adjusted depending upon the relative activity of the material as shown in Example 2 below.
• histidine compounds for administration in the compositions and for the method of this invention I prefer L-histidine HCLH O.
• This compound is very palatable in apple juice and the loading dosages dissolve readily in the amounts of juice described above.
• the free base of histidine is very poorly soluble.
• MMa precursors useful in the method of this invention are DL-valine, L-valine, DL-threonine, thymine and DL-isoleucine, in general order of preference.
• DL valine is preferred.
• L-valine has the advantage.
• DL-threonine is a preferred compound despite its lower activity level because it is very soluble and is relatively tasteless, having only a slightly sweet after taste.
• Thymine is relatively inexpensive and may under certain circumstances oifer advantages as it is not an amino acid.
• DL- isoleucine is included, despite its lower patient acceptance, because of its high activity level.
• MMa precursors alone, in loading dosages and manner of administration described, and collection of urine for a period of 24 hours from the initiation of the loading dosage procedure, followed by the assay of an aliquot of such urine for its methylmalonate content, provides a specific test for the identification and determining the severity of vitamin B deficiency according to the criteria specified herein and illustrated in the examples which follow.
• Urine from the test subjects is collected in suitable clean containers to which sufiicient acid (usually HCl) is added to keep the pH of the final volume below 2.0.
• sufiicient acid usually HCl
• the MMa and FIGLU in the urine is stable for weeks at room temperature.
• an appropriate aliquot of urine is filtered or centrifuged to remove any sediment, and the pH is adjusted appropriately for the FIGLU or MMa assay.
• the volume of the total collection of urine is measured and recorded. Preservatives may be added if they are found not to interfere with the particular assay methods.
• Enzymatic assays (2) Microbiological assays (3) Chromatographic assays (4) Chemical assays These assays have been outlined and described in the following reference: Luhby and Cooperman, Advances in Metabolic Disorders, vol. 1, pp. 263 to 334, Academic Press, New York, N.Y., 1964.
• each of these can be used for estimating the presence and amount of certain concentration ranges of FIGLU in urine and other body fluids.
• the enzymatic assays presently available are preferred for the estimation of FIGLU in urine.
• One such assay is the hog liver FIGLU transferase-cyclodeaminase enzyme (T-C) assay described in US. Pat. No. 3,157,575 and in Lubby and Cooperman, Advances in Metabolic Disorders, vol. 1, pp. 263 to 334, Academic Press, New York, N.Y., 1964.
• T-C hog liver FIGLU transferase-cyclodeaminase enzyme
• the aforementioned sources provide the details of the preparation of the reagents, handling of the unknown sample, the method for carrying out the assay and calculating results. This is the preferred method because it has the greatest sensitivity of the available assays, being able to detect with confidence as little as 1 to 2 mgm. of FIGLU per ml. per urine.
• FIGLU it is the most specific for FIGLU and is not influenced by in terfering compounds commonly occurring in urine, especially in those patients concerned herein.
• This assay has the further advantage of simplicity of performance, reproducibility of results, and lends itself to determination of FIGLU in multiple specimens in the average clinical laboratory.
• the microbiological assays using Lactobacillus arabinosus as the test organism, have good specificity but are generally too cumbersome for the routine clinical laboratory.
• chromotographic assays have achieved popularity and are readily performed with equipment commonly available in the average clinical laboratory. However, such assays are qualitative and do not readily detect urinary FIGLU at concentrations below to micrograms per ml. In addition, interfering substances may confuse the readings.
• methylmanic acid methylmalonate
• concentration of methylmanic acid can be measured by a variety of techniques which fall into three main classes, vapor phase chromotography, thin-layer chromatography and liquid colorimetry.
• Vapor phase chromotographic techniques are the most sensitive. Those that are useful here involve the solvent extraction of the MMa from the urine and the direct determination of methylmalonate according to the procedure of Hoffman and Barboriak (Anal. Biochem. 18, 10 (1967)), or by determinations on volatile derivatives of methylmalonate (Cox and White, Lancet 2, 353-856, 1962). Experience with these procedures indicates it is desirable to form the volatile derivatives prior to vapor phase chromatography since the native acid is decomposed in and on the column during the procedure.
• the weaklybasic anion exchange resin employed above removes the MMa from the urine onto the resin, it also removes several other diazo-reactive substances which then appear at concentrations of from 25 to 70 mgm. liter as determined at the critical MMa wave length. The presence of these compounds renders less precise the estimation of the urinary MMa by this method, particularly when the latter is in the diagnostically lower ranges.
• a strongly acidic cation exchange resin in the hydrogen phase is employed to treat the eluate from the weakly basic anion exchange resin.
• This strongly acidic cation exchange resin removes most of the interfering substances, while allowing the MMa to pass through thus permitting a more accurate and confident estimation of urinary MMa at levels as low as 20 mgm./ liter of urinary sample.
• Resin I A weakly basic anion exchange resin is used, such as Dowex AG 3 x 4 which has polyalkylamine functional groups attached to a styrene-(4%) divinylbenzene polymer lattice, 200-400 mesh particle size, in the chloride form.
• the resin is washed with ten volumes of triple distilled water and suspended in one to two volumes of distilled water until used. It may be stored in a dark glass bottle at 4 C. for several months.
• Resin II A strongly acidic cation exchange resin is used, such as Dowex, 50 W x 8 which is Composed of nuclear sulfonic acid exchange groups attached to a styrene-(8%) divinylbenzene polymer lattice, 200400 mesh particle size, in the hydrogen form. The resin is washed and stored as above.
• 0.025 to 0.065% p-nitroaniline solutions can be used for measurement of low concentration of methylmalonate (1-100 mg./liter).
• concentrations between 500 and 3000 mg. of methylmalonate a 0.01% solution may be desirable.
• Acetate bufier at pH 4.3 The buffer is prepared by dissolving 8.2 gm. of anhydrous sodium acetate in distilled water and making the solution up to 100 ml. with additional distilled water. The pH is adjusted to 4.3 with acetic acid.
• Methylmalonic acid standards Two methylmalonic acid standards are prepared, one containing 0.005 M and the other 0.01 M methylmalonic acid.
• test subjects urine is filtered or centrifuged to remove sediment and the pH adjusted to 6.5 with moderating strong alkalis.
• a suspension of Resin II is introduced into another 1 x 20 cm. glass column and lightly packed to a depth of 4 cm. by gentle air pressure.
• the efiiuent is then adjusted to pH 1.1 with 8 N NaOH.
• the color developed is then read in a spectro photom eter as 620 millimicrons employing suitable blanks and a methylmalonic acid standard.
• Each set of determinations is accompanied by a reagent blank containing 1.0 ml. 0.1 N HCl, 1.5 ml. diazo reagent, 1.5 ml. acetate buffer; and two methylmalonic acid standards:
• the first MMa standard contains 0.05 ml. of 0.005 M, the second standard 0.05 ml. of 0.01 M methylmalonic acid. each added to 0.95 ml. 0.1 N HCl, in separate tubes, to which 1.5 ml. diazo reagent and 1.5 acetate bufier are added. These are then treated from this point in the manner described for the sample above.
• optical density (O.D.) of the standard, the reagent blank and the unknowns are read in 1 cm. light path cuvettes in a spectrophotometer at 620 millicrons.
• the concentration of methylmalonic acid per liter of urine in normal control subjects by this method has been less than about 30-40 mg.
• EXAMPLE 1 The following experiments illustrate the efiect of methylmalonate precursors and histidine, administered alone and in various loading dosage combinations, upon urinary methylmalonate and formiminoglutamic acid excretion in vitamin B deficient, folic acid deficient and normal subjects.
• Experiment B illustrates 24 hour urinary MMa augmenting action of DL-threonine when given alone and when given in combination with histidine.
• the test of Experiment B, day 6, shows the decreased but still considerable activity of DL-t-hreonine when given together with histidine.
• the test of Experiment C, day 4, illustrates Urine Excretion e/ y) Total Dose Exper Compound loading administ.
• MMa FI GLU Day administered dose g. method total A total Experiment A 1 None 200 2.5 2 DL-valine 20 6 816 616 3.0 3 None 210 2.
• 205 H 16.0 5 None 215 3. 2 6 L-histidine DL-valine +20 5g.+6.7g. x 3 q. 411 440 225 16.9
• Tests of Experiment C, day 6 and day 8 illustrate the differential diagnostic value of combinations of DL-isoleucine and histidine and thymine and histidine, where in each of the latter combinations produced a striking increase of the 24 hour urinary MMa output, but no significantly diagnostic increase of 24 hour urinary FIGLU, signifying in each instance, the presence of vitamin B deficiency but not folic acid deficiency.
• Subject B 'Female, age 36 years, lbs. body weight.
• Diagnosis Pernicious anemia, treated (early relapse).
• Clinical data No complaints Hgb. 13.2 gms. percent, RBC 3.68 milli0n/mm. mean RBC corpuscular volume 98, absent gastric intrinsic factor, serum vitamin B activity level 205 /.L,lLg-/II11., serum folic acid (L. casei) activity level 8.1 mag/ml marrow cytology equivocally megaloblastic.
• Loading dosages were administered orally, and urine collected in the manner described in text. Symbols similar to those above.
• Subject C Female, age 57 years, 115 lbs. body weight. Diagnosis: Sprue malabsorption syndrome. Clinical data: Weakness, pallor, diarrhea. Hgb. 7.2 gms percent, RBC 2.2 million per/mm. mean RBC corpus- 3O cular volume 102M, normal gastric intrinsic factor, markedly reduced xylose absorption, serum vitamin B intestinal absorption of xylose, serum vitamin B activity level 360 tg/ml, serum folic acid activity (L. casei) 3.0 m g/ml, marrow cytology megaloblastic 4+.
• Loading dosages were administered orally, and urine collected in the manner described in text. Symbols similar to those above.
• Loading dosages were administered orally, and urine collected in the manner described in text. Symbols similar to those above.
• Clinical data Weakness and pallor Hgb. 6.3 gms percent, RBC 1.9 per cu mm., mean RBC corpuscular volume 106 cu. ,u, normal gastric intrinsic factor, normal gastro- FOLIO ACID DEFICIENCY, MILD Urine excretion, mg./day Total loading MMa incre- FIGLU dose, g. Administration method total ment L-histidine. HCLHZO, given throughout above.
• Sub ect F Loading dosages were administered orally, and urine collected in the manner described in text. Symbols similar Female, age 28, 120 lbs., 7 months of gestation. to those above.
• Clinical data Pallor. Hgb. 8.2 g. percent, RBC 2.1 mil- EXAMPLE 3 lion per cu. mm, marrow cytology, megaloblastic 4+. Values in normal subjects: The range of 24 hr. urinary FOLIO ACID DEFICIENCY, MODERATELY SEVERE Urine extelretion, mg. ay Total loading MMa Incre- Exper. Day Compound administered dose, (g.) Administration method total ment FIGLU 1 None 5.6 2 DL-valine. 20 6.7 g. x 3 q.4 h 4.
• a method for the dilferential diagnosis of folic acid and vitamin B deficiencies in humans which comprises administering to the diagnostic subjects loading dosages of:
• valine L-isoleucine, DL-isoleucine, L-threonine, DL-
• said loading dosages aggregating, during a loading period, from about to about 20 gm. of (a) and from about to about 100 gm. of (b); collecting the urine of the diagnostic subjects during a period of about twenty-four hours from the commencement of said loading period; and determining from said collected urine the respective concentration levels and total amounts of formiminoglutamic acid and methylmalonate.
• methylmalonate precursor is the racemate of an amino acid selected from the group consisting of valine, isoleucine, threonine, homoserine and the non-toxic salts thereof.
• composition for the differential diagnosis of folic acid and vitamin B deficiences which consists of:
• composition according to claim 7 wherein said precursors of methylmalonate are in the form of nontoxic salts thereof.
• composition according to claim 7 wherein said loading dosages are contained in a liquid vehicle and in which each loading dose is contained in about 8-32 fluid ounces of said vehicle.
• composition according to claim 9 wherein the concentration of (b) in said vehicle is about 0.5 to about 10 times the concentration of (a).
• composition according to claim 9 wherein said vehicle is apple juice.
• composition according to claim 13 wherein the methylmalonate precursor is DL-valine.
• composition according to claim 7 wherein the methylmalonate precursor is DL-threonine.
• composition according to claim 7 wherein the methylmalonate precursor is thymine.
• composition according to claim 7 wherein the amount of (a) as L-histidine hydrochloride.H O is from about 1.7 to about 6.6 gm. per unit dose and the amount of DL-valine is from about 5 gm. to about 20 gm. per unit dose; three of said unit doses aggregating a loading dose as set forth in claim 7.
• composition according to claim 7 wherein the methylmalonate precursor is DL-isoleucine.
• composition according to claim 7 wherein the methylmalonate precursor is the racemate of an amino acid selected from the group consisting of valine, isoleucine, threonine and homoserine and the non-toxic salts thereof.
• the method for the determination of vitamin B deficiency states in humans which comprises administrating to diagnostic subjects loading dosages of methylmalonate precursors selected from the group consisting of thymine and the racemates of valine, isocleucine, threonine and homoserine and the non-toxic salts and mixtures thereof, said loading dosages aggregating, during a loading period, from about 10 to about gm.; collecting the urine of the diagnostic subjects during a period of about 24 hours from the commencement of said loading period; and determining from said collected urine the concentration level and total amount of methylmalonate.
• methylmalonate precursors selected from the group consisting of thymine and the racemates of valine, isocleucine, threonine and homoserine and the non-toxic salts and mixtures thereof, said loading dosages aggregating, during a loading period, from about 10 to about gm.; collecting the urine of the diagnostic subjects during a period of about 24 hours from the commencement of said loading period; and
• composition for the differential diagnosis of folic acid and vitamin B deficiencies which consists of:

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• 1968
  • 1968-02-21 US US707303A patent/US3577511A/en not_active Expired - Lifetime
• 1969
  • 1969-01-23 IL IL31480A patent/IL31480A/xx unknown
  • 1969-01-24 IE IE98/69A patent/IE33039B1/xx unknown
  • 1969-01-31 GB GB5435/69A patent/GB1219884A/en not_active Expired
  • 1969-02-10 BE BE728191D patent/BE728191A/xx unknown
  • 1969-02-11 DE DE19691906743 patent/DE1906743A1/de active Pending
  • 1969-02-12 BR BR206347/69A patent/BR6906347D0/pt unknown
  • 1969-02-13 ES ES363611A patent/ES363611A1/es not_active Expired
  • 1969-02-17 FR FR6903907A patent/FR2002311A1/fr not_active Withdrawn
  • 1969-02-20 NL NL6902675A patent/NL6902675A/xx unknown
  • 1969-02-21 CH CH265969A patent/CH513638A/de not_active IP Right Cessation

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