US3577511A - Process and compositions for differential diagnosis of the megaloblastic anemia syndromes - Google Patents

Abstract

A METHOD AND SEVERAL COMPOSITIONS FOR THE ACCURATE AND EARLY DIFFERENTIAL DIAGNOSIS OF THE DEFICIENCY STATES CAUSING MACROCYTIC MEGALOBLASTIC ANEMIA SYNDROME IS PROVIDED IN WHICH ONE OF SEVERAL COMBINATIONS OF SPECIFIED AMINO ACIDS OR OTHER SPECIFIC PRECURSOR COMPOUNDS ARE ADMINISTERED TO THE TEST SUBJECT IN METABOLIC LOADING DOSES AND THE URINARY EXCRETION OF CERTAIN UNUSUAL METABOLIC PRODUCTS IS DETERMINED. THE METABOLIC ANOMALY IN FOLIC ACID DEFICIENCY STATES IS FORMINOGLUTAMIC ACID (FIGLU); IN COBALAMIN VITAMIN B12 DEFICIENCY STATES IT IS METHYLMALONATE (MMA). THIS DIAGMOSTIC PROCEDURE DESCRIBED HEREIN IS POSITIVE EVEN BEFORE THE CHARACTERISTIC ANEMIA IS DEVELOPED. THE RESULTS OF THE DIAGNOSTIC PROCEDURE DESCRIBED PERMITS FROM THE ANALYSIS OF ONE URINE SPECIMEN THE ACCURATE AND DEFINATIVE IDENTIFICATION OF THE PRESENCE AND SEVERITY OF A FOLIC ACID AND/OR VITAMIN B12 DEFICIENCY STATE AND THUS AFFORDS A BASIS FOR DETERMINING SPECIFICALLY THOSE TWO DEFICIENCY STATES. OTHER ASPECTS OF THE INVENTION ARE DESCRIBED IN THE SPECIFICATION.

Description

United States Patent Int. Cl. G01n 33/16 US. Cl. 424-9 22 Claims ABSTRACT OF THE DISCLOSURE A method and several compositions for the accurate and early differential diagnosis of the deficiency states causing macrocytic megaloblastic anemia syndrome is provided in which one of several combinations of specified amino acids or other specific precursor compounds are administered to the test subject in metabolic loading doses and the urinary excretion of certain unusual metabolic products is determined. The metabolic anomaly in folic acid deficiency states is formiminoglutarnic acid (FIGLU); in cobalamin vitamin B deficiency states it is methylmalonate (MMa). This diagnostic procedure described herein is positive even before the characteristic anemia is developed. The 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.

My previous patent, US. Pat. 3,157,575, issued Nov. 17, 1964, describes the administration of metabolic loading dosages of the amino acid, histidine, and the measurement of the subsequent excretion level and amount of formiminoglutamic acid (FIGLU) into the urine, to specifically and accurately identify the presence and severity of the folic acid deficiency syndrome. Because over 98% of the megaloblastic anemia syndromes in humans is caused by either a deficiency of folic acid or cobalamin or a combination thereof, this method, by specifically identifying folic acid deficiency, permitted a partial diagnostic separation of the syndromes associated with the megaloblastic anemias. The red blood cell and certain other histologic characteristics of these syndromes are morphologically identical under the microscope.

My prior method positively identified only the syndrome and anemia caused by folic acid deficiency. The diagnosis of cobalamin deficiency could only be made inferentially.

The term 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.

The term 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.

It is of importance that a positive diagnosis of each type of deficiency leading to the syndrome characterized in its overt form by macrocytic megalobl-astic anemia, be established. Therapy for each deficiency state is different and must be specific. Administration of the non-deficient of these two vitamins to a subject deficient of the other, while temporarily clearing the blood abnormality, may mask other symptoms of the deficiency state and lead to serious consequences for the patient.

For example, administration of 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. Cases have been reported in which the administration of folic acid to cobalamin deficient patients has appeared to precipitate an explosive, progressive and severe form of the neurological lesions of Addisonian anemia while presenting a picture, based on blood counts and bone marrow findings, of hematopoietic improvement. Since the initial neurological symptoms of the cobalamin deficiency underlying and leading to pernicious anemia are generally diffuse and nonspecific, the improving or improved hematopoietic picture can be misleading as to the true vitamin deficiency underlying the patients disorder, and through a resulting confusion, delay the institution of the proper specific therapy to a point where the progression of neurological lesions may lead to irreversible changes. 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.

Similarly, a positive true and definitive diagnosis of folic acid deficiency state must be made or the administration of theraputic dosages of the unneeded cobalamin (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. If the folic acid deficiency is severe, no improvement may occur to vitamin B therapy even in high dosage, permitting the folic acid deficiency to become progressively more severe With a resulting Worsening anemia, exposing the patient to possibly serious and even fatal hemorrhage from the thrombocytopenia and fulminating and intractable infection induced in part by the resulting neutropenia of the severe folic acid deficiency state.

Because of the previous lack of a clinically simple and convenient method for the positive differential diagonsis of the specific deficiency state causing the macrocytic megaloblastic anemia syndrome, some physicians in the past have prescribed hematinics to be taken orally and containing both of these agents. This practice is not only expensive but may lead to the same serious consequences in unrecognized pernicious anemia patients or may obscure the presence of other underlying disease.

As a result, the US. Food and Drug Administration has interdicted and severly restricted folic acid from vitamin preparations which are available to the public without a doctors prescription.

It is an object of this invention to provide a differential diagnostic method for identifying the persence and severity of the specific folic acid and or vitamin B deficiency state associated with the magaloblastic anemia syndromes in incipient or fully developed states.

It is an object of this invention to provide a combination of materials for this differential diagnostic method which simply, uniquely, quickly and positively differentiates the folic acid from the cobalamin (vitamin B deficiency syndrome of megalobastic anemias in humans, and identifies the presence of either folic acid and vitamin B deficiency or both.

It is a further object of this invention to provide a method for diagnosis of disturbances in amino acid and/ or fatty acid utilization and metabolism which disturbances prevent the proper utlization of certain specific amino acids and/ or fatty acids by the body, causing anomalies in the excretion of specific identifying chemicals, said distrubances resulting from deficiencies of folc acid and/ or cobalamin (vitamin B Other objects and advantages of this invention will become apparent from the detailed description and examples which follow.

THE INVENTION 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. I have found that when loading doses, of histidine and MMa precursor are administered to subjects with folic acid and/ or vitamin B deficiency, 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. Normal individuals as well as those with other types of anemia, do not excrete increased amounts of these substances when loading materials are administered in the manner described in this invention. The determination of the amount of these substances excreted is preferably made upon the pooled urine collected during the 24 hour period following the initiation of the loading dosages.

I have further found that 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. Of these 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.

I have also found unexpectedly, that 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. Preferably the loading dosages should be administered in three portions at 4 hour intervals.

For adults, for example, 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. For maximum FIGLU and MMa outputs at a particular loading dose level, 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.

In addition to the novel method which provides for 4 the concurrent administration of the histidine and the MMa precursor, this invention also includes compositions for the simultaneous administration of these materials in specified dosages and proportions.

The 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. However, surprisingly, when the racemates of the amino acids are administered, 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.

I have found, also, that the amount of most of the MMa precursors, when administered with histidine, is different, for some unexplained reason, from the amount necessary for its administration when the MMa precursor is given alone to enhance MMa output. The amount of MMa precursor in my combination was therefore increased above the level that might be inferred from the MMa excretion induced by the single agent administration to obtain sufficient loading to insure positive diagnosis.

Loading dosage, as the term is used herein, 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.

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. On an equimolar basis 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.

Efforts to test the enhancing effect of propionate and homocysteine on urinary MMa excretion in vitamin B deficiency which have been reported as active MMa precursors were regularly frustrated in humans by nausea, followed often by vomiting, despite the administration of these substances in a variety of menstrua, dose schedules and pH levels.

In other experiments, equimolar mixtures of L-valine and L-isoleucine; L-valine and L-threonine; and L-valine, DL-isoleucine and DL-threonine, do not produce urinary MMa excretions significantly different from those which might be expected from the equimolar administration of the individual amino acids.

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. No significant urinary MMa excretion occurs in vitamin B deficiency after D-valine, L-methionine, DL-serine, L-asparagine, L-aspartic acid, beta-alanine or L-histidine, when administered in approximately equimolar amounts to those of the previously mentioned components.

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.

I have found, further, that when the level of FIGLU in the 24 hour pooled urine following administration of a member of the group FIGLU precursors, in the loading dosage and procedure described herein, is in excess of about 30-35 micrograms per liter and is excreted in an amount in excess of about 35 mgm. during the 24 hours, from the initiation of the loading procedure, such values are diagnostic for the presence of folic acid deficiency states. Further when the levels are below these indicated amounts, such levels signify the absence of such a deficiency state. In general, the degree of increased excretion parallels the degree of folic acid deficiency.

Similarly when the urinary MMa in this 24 hour pooled urine sample, following initiation of loading, shows that the MMa is excreted in amounts in excess of about 30 mgm. per 24 hour period, such amounts signify a vitamin B deficiency state. Urinary excretion of less than about 30 mgm. Within the 24 hours after initiation of the loading dosage, according to the method of this invention, signifies the absence of such a deficiency state. In general, the degree of increased MMa excretion parallels the degree of vitamin B deficiency.

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.

With regard to the 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.

Among the 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.

Among the 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. On the basis of cost and activity DL valine is preferred. On the basis of activity alone, 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. Because of the unexplained and unforeseen increased activity in the racemic amino acid on an equivalent molecular basis to the L-form, as MMa precursors, and because the racemates are considerably cheaper than the L-form, which until now was the only form of these amino acid MMa precursors shown to be active, these forms are most suitable for diagnostic use in general clinical practice.

The invention in its various ramifications is exemplified in the procedure section and the examples which follow. The procedures described are useful with details being given for a novel variation for determining methylmalonate. The method for the detremination of formiminoglutamic acid is not set forth in detail as it is contained in the included references.

Further, use of the 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.

The examples illustrate cases wherein megaloblastic anemic subjects, having given informed consent, were given loading dosages of the components and the combination of this invention in order that the parameter and safety of the procedure be explored. Detailed laboratory findings, providing the basis for diagnosis and for recom mending specific formulations and proportions can be seen in the reported data. Further, experience with a general hospital and outpatient otfice population, wherein these tests were run on patients suspected of having these metabolic deficiencies, has shown the general reliability and specific applicability of the practice of this invention.

Experience with the combination of this invention, which has been administered in several hundred loading dosage tests followed by assays, indicates the validity of this differential diagnostic method and its associated loading dosage composition. In no case, within the recommended loading dosage ranges of the various combinations described as effective, was there any false positive result; and no false negative results were encountered.

While all of the assays have been performed according to the preferred procedure, this invention is not limited to these procedures since any sufiiciently accurate method for assaying FIGLU and MMa should lead to comparable diagnostic results.

Since all the compounds are normal components of food they are safe for oral administration. Moreover, my experience with the compounds alone and in combination has given rise to no contraindication for their ad ministration according to this invention. This experience includes subjects ranging from infants, pregnant women and subjects in various stages of folic and vitamin B deficiency states.

SAMPLE COLLECTION 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.

Under these conditions, the MMa and FIGLU in the urine is stable for weeks at room temperature. Just prior to assay, 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.

ASSAY PROCEDURES While the presently preferred determination of FIGLU and MMa are performed on urine specimens, it is possible to perform such determinations, by variations of the methods set forth below on blood plasma and other body fluids where these deficiency indicator substances appear in greater than normal concentrations after administration of loading dosages of the precursor compounds described therein.

METHODS FOR THE ASSAY OF FIGLU Several methods for measurement of the presence and concentration of FIGLU have been developed. These methods fall into the following general classes:

(1) 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.

With suitable modifications 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. 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. In addition, 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.

Two other enzymes methods have been described including one by Silverman et al. (J. Natl Cancer Inst., 20, 71, 1958) which uses a microbiological assay for determination of the end product, and a modification of the latter by Chanarin-Bennet (Brit. Med. 1., vol. 1, pp. 27 and 985, 1962) which converts the end product to a substance measurable spectrophotometrically.

All three of these assays are sufficiently sensitive to be used for detecting the diagnostically important ranges of urinary FIGLU concentrations. However, the T-C enzyme assay, as noted, is preferred. This enzyme method, set forth in detail in the aforementioned patent, was used for the determination of the FIGLU data reported in the appended clinical examples.

The microbiological assays, using Lactobacillus arabinosus as the test organism, have good specificity but are generally too cumbersome for the routine clinical laboratory.

A number of 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.

Chemical assays for FIGLU are available. Two such have been described in the reference cited earlier. These assays although simple to perform lack suflicient specificity and sensitivity to be presently useful for determinating FIGLU in urine in patients with which this invention is concerned.

METHODS FOR THE ASSAY OF METHYLMALONATE The concentration of methylmanic acid (methylmalonate) 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 assay procedures which are based on solvent extraction of urinary MMa and the determination of its concentration by vapor phase chromotography, while they are the most sensitive and specific assays presently available, are cumbersome, lengthy and require both special apparatus and highly trained personnel for the use of the apparatus and interpretation of the resultant data.

A method combining solvent extraction of the MMa from urine with thin layer chromotography of the residue has been described (Hinterberger, B-ashir and Jones, Proc. Australian Assoc. of Clin. Biochem., 1, 143, 1965). The procedure is essentially qualitative and satisfactory modifications for the quantitative aspects of this invention have not as yet been perfected. Other methods under study involve molecular sieves chemical charges, enzyme microbiological conversions, electron capture methods and liquid phase chromotography.

The other main group of methods which are more useful for the average clinical laboratory are based on colorimetric assays. An excellent method for the determination of urinary MMa has been suggested by Giorgio and Plant (J. Lab. Clin. Med. 66, 667-676, 1965). This method concentrates the methylmalonate on a weakly basic anion exchange resin from which it is eluted and coupled with diazotized p-nitroaniline. At high pH, an emerald green color develops, with a molar absorbency peak of about 10,000 at 620 m wave length. Experience has shown the absorbency to be linear over a concentration range of 0.6 to 12 ,ugm. per ml. which under the conditions suggested by the authors provides a determination range of MMa concentrations of from 12 to 240 mgm./ liter of urine.

Although 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.

In order to overcome this problem, I prefer to use a modification of the Giorgio and Plant procedure whereby a second ion exchange resin, 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.

A detailed description of my preferred clinical analytical procedure for MMa follows.

MATERIALS AND REAGENTS (1) Resin columns:

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.

(2) p-Nitroaniline: A 0.075% solution is prepared by dissolving 375 mg. of recrystallized reagent grade pnitroaniline in 500 ml. of 0.2 N hydrochloride acid. The solution may be stored in a dark glass bottle and remains stable for many months at room temperature.

Alternatively, for measurement of low concentration of methylmalonate (1-100 mg./liter), 0.025 to 0.065% p-nitroaniline solutions can be used. For concentrations between 500 and 3000 mg. of methylmalonate, a 0.01% solution may be desirable.

(3) 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.

' (4) 0.5% sodium nitrite: For lower concentrations of the p-nitroaniline reagent, 0.20 to 0.45% NaNO may be desired.

(5) 0.2 M sodium acetate.

(6) 3 N sodium hydroxide.

(7) 8 N sodium hydroxide.

(8) 0.1 N hydrochloric acid: Adjust to this pH 1.1, if necessary.

(9) Preparation of diazo reagent: 4.0 m1. of the 0.5% NaNO reagent is added to ml. of the 0.075% pnitroaniline reagent at room temperature. The mixture is cooled in an ice bath to 4 C. and 4.0 m1. of cold 0.2 M sodium acetate reagent is added. The resulting diazo reagent is stable for 24 hours at 4 C.

(10) Methylmalonic acid standards: Two methylmalonic acid standards are prepared, one containing 0.005 M and the other 0.01 M methylmalonic acid.

DETAILS OF ASSAY PROCEDURE Resin column preparation and treatment of sample: A suspension of Resin 1 is placed in a 1 x cm. glass column with a sintered glass plug and packed lightly with gentle air pressure to a depth of 1 x 2.5 cm. It is preferred that the column depth of the resin does not exceed 2.7

An appropriate sized aliquot of the test subjects urine is filtered or centrifuged to remove sediment and the pH adjusted to 6.5 with moderating strong alkalis.

A 5.0 ml. aliquot is allowed to flow by gravity through the column. The resin is then washed two times with 50 ml. portions of distilled water. The methylmalonic 10 acid is then eluted from hte resin by gravity flow with 20.0 ml. of 0.1 N HCl.

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. To 10 ml. of the above eluate from Resin I, is added 1.0 ml. of concentrated HCl, and the solution placed on the Resin H column and allowed to flow through by gravity. The efiiuent is then adjusted to pH 1.1 with 8 N NaOH.

Color development: In 13 x 75 mm. glass tubes, 1.0 ml. of the acidified efliuent from the Resin II column above is added to 1.5 ml. of 1.0 M acetate buffer (pH 4.3). 1.5 ml. of cold diazo reagent is then added, the contents mixed and the mixture heated for 3.0 minutes in a wtaer bath at -94 C. 1.0 ml. of 3.0 N NaOH is then added to the hot mixture. The tubes are stoppered, the contents mixed, and the tubes are removed from the water bath and allowed to cool at room temperature for 10 minutes.

For concentrations of expected urinary MMa above 240 mg./liter, an appropriately smaller amount of the acidified efiluent than 1.0 ml. is employed; the diflerence in volume being made up with 0.1 N HCl.

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.

The 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.

Under standard conditions of assay, the following formula is used to calculate the concentration of methylmalonic acid per liter of urine:

O.D. sample-OD. reagent blank O.D. standard-OD. reagent blank 1000 volume eflluent urine volume effluent aliquot vol.

mg. MMa/liter urine X (standard X 1 See the following equation:

0.D. standard (corrected):

O.D. 0.005 M standard (corrected) 2+O.D. 0.1 standard (corrected) 2' mg. MMa/liter urine:

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.

1 1 Subject A VITAMIN B12 DEFICIENCY SEVERE with megaloblastic anemia due to B deficiency but not folic acid deficiency wherein the loading dosages are given at the same time and the determination of the urinary MMa and FIGLU content is made from a single urine collection. In the experience with subject B below, the value of the specific diflerential diagnostic aspects of this invention is illustrated where the baseline endogenous 24 hour urinary MMa excretion is not diagnostic for B deficiency.

The tests in Experiment B illustrate the 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. 4 L-llistidine 15 g. x3q. 4b.". 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

Experiment B 1 None 180 2. 0 2 DL-threonine 354 174 2. 2 3 None 185 3.2 4 L-histidine 15 5g.x3q.4h 200 17.0 5 None 195 4. 5 6 DL-thrconine+L-histidine -1-15 6.67+5 g. x 3 q. 4h 280 16. 5

Experiment 0 l None 190 3.4 2 DL-threonine 30 10 g.x3q.4h 630 440 4.6 3 None 200 2.0 4 DL-threonine+L-histidine 30+15 10 g. +5 g. x 3 11.4 h 22.8 5. l 6- 15-1-20 5 g.+6.7 g. x 3 q.h 1,00 200 18. 7 7 None 180 4.3 L-h' tidine* thymine 15 *10 5g. *3.3x3q.4h 360 170 16. 1

Each precursor given concurrently, hr. after the other, not simultaneously.

This patient subsequently made a classical hematological response to 3 g. vitamin B intramuscularly daily for 3 days, confirming in this manner the presence of vitamin B deficiency (Luhby and Cooperman, adv. in Metab. Dis. 1, 263-334, 1964, Academic Press, New York, N.Y.).

In the above practice of the invention, it can be seen that in a vitamin B deficient subject, DL-valine administered alone, produced a striking increase in the urinary MMa excreted during the 24-hour period commencing with the loading dose administration; during the latter period the urinary FIGLU excretion was unaltered; subsequently histidine alone did not significantly affect the 24 hour urinary MMa excretion, and although the 24 hour urine collected from the commencement of histidine loading showed a moderate increase in urinary FIGLU output, this level is below the level which I have found is diagnostic for folic acid deficiency.

In subsequent administration of histidine and DL-valine in combination, the net 24 hour output of MMa in the urine above baseline was in the order of /3 that when the same dose of DL-valine was given alone. Nevertheless, the increase of MMa was striking. Although in this patient the high (about 200 mg.) baseline 24 hour urine MMa excretion is indicative of vitamin B deficiency, the test of experimental day 6 illustrates how the invention opcrates as a specific differential diagnostic method to furthe! identify the specific B deficiency state in a subject how the reduced efiec't of giving an amino acid MMa precursor simultaneously with histidine can be overcome and provides an example of the data basis for the different and increased proportional loading dosages recommended in the combinational aspects of the invention with a FIGLU precursor, as compared to the use of the MMa precursors alone. 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.

VITAMIN B12 DEFICIENCY MILD Urine excretion,

mg. 2. 7 Total loading MMa FIGLU Exper.Day. Compound administered dose, g. Dose Admirdst. Method total A total Insignficant rise.

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.

FOLIO ACID DEFICIENCY, MODERATELY SEVERE Urine excretion,

Total mg./day loading Exper. dose, MMa Incre FIGLU Day Compound administered g. Administration method total ment 7. 2 15 5 g. x 3 q 230 8. 20 6.67g.x3q.4h 6.0 None 5. 2 L-valine+L-histidine- 20+15 6.67 g.+ g. x 3 q. 4 h 220 one 5. 6 RL-isoleuoine-l-L-histidine 20+15 6.67 g.+5 g. x 3 q. 4 h 234 one 4.1 L-threonine+L-histid1ne 20+15 6.67 g.+5 g. x 3 q. 4 h 242 one 8. 7 Thymine+L-histidine 10+15 3.3 g.+5 g. x 3 q. 4 h 229 *L-hlSlZidlIlG-HCLHzO, given throughout above.

activity level 110 tg/ml, serum folic acid (L. casei) Subject B activity 2.8 mpg/ml, marrow cytology megaloblastic 50 Male, age 7 years, 1601bs body Weight Diagnosis: Nutritional macrocytic anemia.

COMBINED VITAMIN B 2 AND FOLIO ACID DEFICIENCY; MODERATE FOLIO ACID, MILD B12 DEFICIENCY Urine excretion,

mgJday Total loading MMa Fl GLU Exper. Day. Compound administered dose, g. Dose Administ. Method total A total 1 None- 5. 2 2 Lhistidine 22 5.7 Nnnp 20 6. 2 Dlrvaline 97 77 5. 9 None--- 25 4. 1 DL-va 57 32 140 on 22 6. 1 Dlrvaline L hbtldlfle 15 g. 5 g. x 3 q. 4 h 85 63 126 None 20 4. 7 DL-threonine L-histidine-. +15 20 g. 5 g x 3 q 75 55 *L-histidineGHLHzO, given throughout above.

Loading dosages were administered orally, and urine collected in the manner described in text. Symbols similar to those above.

Subject D Male, age 47 years, 145 lbs. body weight Diagnosis: Nutritional megaloblastic anemia.

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.

Diagnosis: Macrocytic anemia of pregnancy.

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. 2 3 5.7 4 12s 5 6.7 6 DL-valine 118 Subject G MMa output in normal adults (20 cases), pregnant Male age months lbs women (18 cases), and infants (12 cases), without load- 7 i I l Diagnosis; Coeliac disfiaw mg, was 0 to 27 mgm. After loading with all of the MMa Clinical data: Pallor. Hgb. 7.0 g. percent, RBC 2.1 milprecursors listed throughout the examples above, alone lion mm}, marrow cytology, megaloblastic equivocal. and in the various doses and combinations with histidine,

FOLIO ACID DEFICIENCY, MILD Urine excretion,

mg. day Total loading MMa Incre- Exper. Day Compound administered dose, (g) Administration method total ment FIGLU 1 None 8.0 2 DL-valine 3.0 1.0 g. x 3 11.4 h 3 4 100 5 6 DL-valine 90 including doses of MMa precursors listed in the earlier EXAMPLE 2 table in the text, did not give urinary MMa excretion Total fi $7355 above 30 mgm. per day, in 320 loading tests. loading Administm. The range of 24 hr. urinary FIGLU excretion in Compound (158, E- 1011 method FIGLU normal adults, (1510 cases), pregnant women (680 cases), 5 2.1 infants and children (420 cases), without loading, was i8 0 to 6 mgm. per days. After loading with all of the MMa 15 1. precursors listed throughout the examples above, alone lg and in various combinations with themselves, did not pro- 10 4:6 duce a 24 hr. urinary FIGLU output greater than 6 mgm. 2% if in 264 loading tests. The various MMa precusors when Don 30 2.0 given in various dose combinations with histidine did not fii g i8 513 produce a 24 hr. urinary PjIGLU output greater than 28 alloisoleucine. mgm. per day, 1n 362 loading tests. 3 Histidine given as the preferred compound, loading Do 10 3.6 dosage and method of administration illustrated and def f f 38 it, scribed herein in vitamin M deficient patients, has not Th Do i8 resulted in a 24 hr. urinary FIGLU output greater than 35 nt -fiiii'gsniiijjji 20 218 mgm. per day after testing over 300 such subjects with L-mgthiOnineHU i8 5-; pure B deficiency. Loading with the various MMa DIrsei r1 e:: 20 217 precursors in over 35 patients with pure folic acid defir rgg ii g %g ciency, has not resulted in an increase of the 24 hr. L-higtidine 116111110 15 1314 urinary MMa output over 30 mgm., in 116 loading tests. xgqfilh 0 While the invention has been illustrated by the foregoing examples with specific procedures and selected llhcremhntebove baseline subjects, it will be apparent that various equivalent changes and modifications may be resorted to or necessary, in carrying out this invention without departing from the scope and spirit thereof. It will be understood that such equivalents are within the purview of the appended claims.

I claim:

1. 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:

(a) a member of the group consisting of histidine and non-toxic salts thereof, and

(b) at least one precursor of methylmalonate selected from the group consisting of thymine, L-valine, DL-

valine, L-isoleucine, DL-isoleucine, L-threonine, DL-

threonine, L-homoserine and DL-homoserine, 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.

2. The method according to claim 1 wherein the precursors of methylmalonate are administered as non-toxic salts thereof.

3. The method according to claim 1 wherein the loading dosages are administered in at least three divided portions within a twelve hour period.

4. The method according to claim 1 wherein the nontoxic salt of histidine is L-hiStidineHCLH O and the methylmalonate precursor is DL-valine.

5. The method according to claim 1 wherein in the 24 hour urine obtained from the commencement of the loading period, a urinary formiminoglutamic acid level in excess of about 30-35 micrograms per ml. and in excess of about 35 milligrams per 24 hours identifies folic acid deficiency, a urinary formiminoglutamic acid level below said values signifies the absence of folic acid deficiency; a urinary methylmalonate level on the same urine in excess of about 30 milligrams per 24 hours identifies vitamin B deficiency, a urinary methylmalonate level below said values signifies the absence of vitamin B deficiency.

6. The method according to claim 1 wherein the 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.

7. The composition for the differential diagnosis of folic acid and vitamin B deficiences which consists of:

(a) a member of the group consisting of histidine and non-toxic salts thereof, and

(b) at least one precursor of methylmalonate selected fiom the group consisting of thymine, L-valine, DL- valine, L-isoleucine, DL-isoleucine, L-threonine, DL- threonine, L-homoserine and DL-homoserine, in loading dosages for adults aggregating from about 5 to about 20 gm. of (a) and from about 10 to about 100 gm. of (b).

8. The composition according to claim 7 wherein said precursors of methylmalonate are in the form of nontoxic salts thereof.

9. The 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.

10. The 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).

11. The composition according to claim 9 wherein said vehicle is apple juice.

12. The composition according to claim 7 wherein (a) is L-histidine.HCl.H O.

13. The composition according to claim 7 wherein the methylmalonate precursor is DL-valine.

14. The composition according to claim 7 wherein the methylmalonate precursor is DL-threonine.

15. The composition according to claim 7 wherein the methylmalonate precursor is thymine.

16. The 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.

17. A composition according to claim 7 wherein the methylmalonate precursor is DL-isoleucine.

18. The 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.

19. 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.

20. The method according to claim 19 wherein the loading dosages are administered in at least 3 divided portions within a 12 hour period.

21. The method according to claim 19 wherein in the 24 hour urine obtained from the commencement of the loading period, a urinary methylmalonate level in excess of about 30 milligrams per 24 hours identifies vitamin B deficiency and a urinary methylmalonate level below 30 milligrams signifies the absence of vitamin B deficiency.

22. The composition for the differential diagnosis of folic acid and vitamin B deficiencies which consists of:

(a) a member of the group consisting of histidine and non-toxic salts thereof, and

(b) at least one precursor of methylmalonate selected from the group consisting of thymine, L-valine, DL- valine, L-isoleucine, DL-isoleucine, L-threonine, DL- threonine, L-homoserine and DL- homoserine, and non-toxic salts thereof in loading dosages for infants and children aggregating from about 0.08 to about 0.14 gm. per pound of body weight of (a) and from about 0.08 to about 1.4 gm. per pound of body weight of (b).

References Cited UNITED STATES PATENTS 3,157,575 11/1964 Luhby 4249 SAM ROSEN, Primary Examiner Page 1 22 8? UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTIGN Patent No. 3 577, 511 D t d May 4-, 197].

Inventor(s) Adrian LQOHard It: is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shox-m below:

r Col. 1, line 63 change "votamin" to vitamin Col. 3, line 4, change "persence" to presence Col. 3, line 6, change "magaloblastic" to megaloblas tic Col. 3, 'line 21, change "distrubances" to disturbances Also in this line, change "folc" to folic Col. 3, line 29, change "deficency" to deficiency Col. 3, line 32, change "loadng" to loading Col. 3, line 34, change "amounts" to amounts Col. 3, line 40, change "vitamn" to vitamin Col. 3, line 41, change "deficent" to deficient Col. 3, line 50, change "L- valne"and"DL- valne" to L- valine and DL- valine Col. 4, line 45, change "dosages" to dosage Col. 5, line 54, change "to" to in Col. 6, line 15, change "in" to of Col. 7, line 40, change "Lubby" to Luhby Col. 8, line 15, change "methylmanic" to methylmalonic Col. 8, line 45, a comma after "sieves" is missing. Also in line 45, "and" after "enzyme" is missing.

Col. 10, line 1, change "hte" to the Col. 10, line 15, change "wtaer" to water Col. 10, line 32, the period after "acid" should be a comma Col. 10, line 38, change "millicrons" to millimicrons Col. 10, line 55, in the formula, change "283.02" to 283.2

Col. 10, line 60, in the formula, change "0. 1" to .OlM

Col. 13, in line with Exp-er. Day 2 of the Table below line 51,

under "FIGLU total", change numeral "5. 7" to read 15.7

Col. 13, line 66, immediately below the Table, change "CHLH O" to o Pa gm 2 UNITED STATES PATENT OFFICE CERTIFICATE OF CORRILC'IIUN Patent No. 3 577 511 Dated May 4, 1971 Inventoflg) Adrian Leonard Luhby It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

COL. 15, first Table, Exper. Day 2', under Administration Method, change "3g. to 5g. 2

Col. 15, line 16, just below the first Table, change "HCLH t0 I-ICl.H 0

Col. 15, second Table, (Subject F) Exper. Day 6, under Administration Method, change "5g. x 4h" to 5g. x 3q. +h

Col. 15, third Table, (Subject G) Exper. Day 6, under Administration Method, change "0.8 x 4h" to 0.8 x 3q.4h

Col. 16, line 56, change "days" to day Col. 16, line 60, change "precusors" to precursors Col. 16, line 66, change "M to B p Col. 18, line 31 (claim 19, line 5) change "isocleucine" to isoleucine Signed and sealed this 12th day of October 1971 (SEAL) Attest:

.FIETCHER JR. ROBERT GOTTSCHALK fi zifig Officer Acting Commissioner of Patents