A n n a l s o f C l i n i c a l L a b o r a t o r y S c i e n c e , Vol. 1, No. 8 Copyright © 1971, Institute for Clinical Science

The Biochemistry of Folic Acid and B12

M. MICHAEL LUBRAN, M.D., P h .D. Harbor General Hospital Torrance, CA 90509

Folic acid and are the pre­ tissues and human red cells as a polygluta­ cursors of coenzymes involved in many mate, typically containing three to seven important biochemical reactions. Most of glutamic acid residues linked by gamma- our knowledge of the biochemistry of these peptide bonds. Human small intestinal has been obtained from the study juice contains the folic conjugase of bacterial metabolism: there have been which is necessary for the hydrolysis and fewer studies of animal or human tissues. absorption of the poly glutamates. Absorp­ The role of these vitamins in man has been tion is an active process that occurs mainly determined mainly by the investigation of in the duodenum and jejunum. patients with or BJ2 deficiency. There Folic acid itself has no biological activity. is still much that is not known about their It is the precursor of a group of coenzymes behavior in man; nor is it certain that all derived from in which the biochemical reactions in which they the N atoms at 5 and 8 and the C atoms participate have been discovered. Reactions at 6 and 7 of the pyrazine ring have been involving folic acid and B12 in micro-or­ reduced. The tetrahydrofolate derivatives ganisms and even animals do not neces­ contain groups attached to N-5, N-10 or sarily occur in man. In particular, megalo­ both atoms by a bridge (table 1). The blastic anemia as seen in the human does coenzyme is attached to its specific enzyme not occur in animals deficient in folate or through the ring. Bi2. The major biochemical reactions of Folic acid is reduced to folic acid and vitamin Bi2 will be described and then to tetrahydrofolic acid by the here with emphasis on those known or be­ enzyme dihydrofolic acid reductase, which lieved to be important in human metabo­ requires pyridine (NADH2 or lism. The literature on the subject is vast. NADPH2). Dihydrofolic acid, which is Recent reviews are cited in the references. the natural substrate, is reduced more readily than folic acid by this enzyme. Folic Acid Folic acid reductases, which convert folic acid to dihydrofolic acid only, have been Folic acid is pteroylglutamic acid (figure described in some . Their role in 1). It consists of a pteridine portion linked man is uncertain. Dihydrofolic acid reduc­ through p-aminobenzoic acid to L-glutamic tase is widely distributed in bacterial and acid. It is found in plants (its name is de­ animal cells. It is strongly inhibited by rived from the Latin folium, a leaf), animal folic acid antagonists such as 236 BIOCHEMISTRY OF FOLIC ACID AND VITAMIN B12 237

Table I. The Tetrahydrofolate H C00H Y '¿;|rCH2-N-

Substituent Formula Attached to N Oxidation level

pteridine ^-aminobenzoic L-glutamic Methyl c h 3 5 Methanol acid acid Methylene c h 2- 5 and 10

pteroic acid Methenyl CH = 5 and 10 Formate V------y------/ Formyl CHO 5 or 10 Formate pteroylglutamic acid (PteGlu) Formimino CH=NH 5 Formate

F ig u r e 1. Structural formula of folic acid. Folate coenzymes include tetrahydrofolate and substituted tetrahydrofolates

(4-aminopteroylglutamic acid) and metho­ the folate coenzymes are derived from the trexate (4-amino- 10-methylpteroylglutamic following compounds: (/3-C) mainly, acid). formate and to a small extent only. Single carbon atoms derived from these Functions of Folic Acid Coenzymes groups are found in: serine (/3-C), formate, The major function of the folate co­ formaldehyde, (C-2 and C-8), is the transfer of one-carbon thymine (5-methyl C) and methionine (5- groups in a variety of synthetic reactions, methyl C). The reactions involved are which depend upon the state of oxidation described below. All are believed to take of the group transferred. The lowest level place in human metabolism. of oxidation is that of methanol (methyl (1) L- serine- interconversion group); next is formaldehyde (methylene Serine hydroxymethyltransferase, tetra­ group); highest is formate (methenyl, hydrofolate and pyridoxal-5-phosphate re- formyl, formimino groups). Coenzymes versibly convert L-serine to glycine. The containing these groups are readily inter­ enzyme is present in human red cells. convertible by specific enzymes, except for CH, OH-CH-COOH + H, PteClu ¡a CH, COOH + 5.10-CH.-H, PteClu 5-methyltetrahydrofolate. The methylene I I NHa NHS level is reduced to methyl by 5, 10- serine tetrahydrofolate glycine methylenetetra- methylenetetrahydrofolate reductase, a hydrofolatc flavo-protein requiring FADH2 (reduced 2) Formimino glutamic acid (FIGlu) con­ flavin dinucleotide); the reverse version reaction does not occur in man. Regenera­ FIGlu is an intermediate in the enzy­ tion of tetrahydrofolate from methyltetra- matic degradation of histidine in animals. hydrofolate is Bj2 dependent and occurs It is converted to glutamic acid by FIGlu during the synthesis of methionine. This formiminotransferase and tetrahydrofolate. process is described in the section on Bi 2. COOH - CH - CH*-CH8 COOH + H4 PteClu ->COOH-CH-CHa-CHa COOH The NADP-dependent enzyme, 5,10-methy- N H N H . I lenetetrahydrofolate dehydrogenase, brings NH = CH formiminoglutamic acid glutamic acid about the interconversion of coenzymes 5-CHNH-iri PtcGlu containing groups at the formaldehyde and formiminotetrahydrofolate formate levels of oxidation. Methenyl and The formiminotetrahydrofolate produced in formyl groups are interconverted by a cyclo- this reaction is converted by cyclodeaminase hydrolase; methenyl and formimino deriva­ into 5,10-methenyltetrahydrofolate; this in tives by a cyclodeaminase. turn is converted by cyclohydrase into 10- The single carbon groups transferred by formyltetrahydrofolate. In patients with 238 LUBBAN folic acid deficiency, the of Syntheses Involving Folic Acid FIGlu is decreased and its excretion in the Coenzymes urine is increased. Excretion is more marked Folic acid coenzymes are involved in the in the presence of a histidine load. Vitamin synthesis of purines, (and thus, B12 deficiency may also give rise to in­ indirectly, in the synthesis of DNA) and creased urinary excretion of FIGlu, but the methionine. The last synthesis also involves increase is not as marked as in folic acid vitamin B]2 and will be described later. deficiency. 1) synthesis 3) Utilization of formate Folic acid is concerned in the introduc­ Tetrahydrofolate formylase (formate-ac- tion of carbon atoms into positions 8 and 2 tivating enzyme), ATP and tetrahydrofolate in the purine ring; different coenzymes are give rise, reversibly, to 10-formyltetrahy- involved. C-8 is introduced by the forma­ drofolate; this coenzyme transfers the tion of formylglycinamide ribonucleotide formyl group to appropriate substrates (for (FGAR) from glycinamide ribonucleotide example, in the synthesis of purines). (GAR) by 5,10-methenyltetrahydrofolate H* PteGlu + HCOOH + ATP *=» l()-CHO-H4 PteGlu + ADP +Pi formyltetrahydrofolate and GAR transformylase (figure 2). Tetra­ hydrofolate is formed. The formyl group of Only 10-formyltetrahydrofolate participates FGAR later condenses with the amide N in formate transfer in purine synthesis. The to form an imidazole ring. energy of hydrolysis of 5-formyltetrahydro- C-2 is introduced by formylation of 5- folate is too low for this transfer to occur. amino-4-imidazole-carboxamide ribonucleo­ This enzyme can be converted into the 10- tide (AICAR) with 10-formyltetrahydro­ formyl form by ATP and magnesium ions. folate and AICAR transformylase to give 4) Formylation of glutamate 4-formamido-5-imidazoecarboxamide ribo­ This takes place through the action of (FAICAR) and tetrahydrofolate glutamate transformylase and 5-formyltetra- (figure 3). AICAR undergoes ring closure hydrofolate; 10-formyltetrahydrofolate is in­ to form inosinic acid, which is subsequently active in this reaction. converted to adenylic, xanthylic and guany- lic acids by appropriate enzyme systems. Glutainate5-CHO-Hi PteGlu formylglutamide -j- II, PteGlu Folate inhibitors (aminopterin, metho­ The reverse reaction is important in for­ trexate) do not inhibit the folate dependent mate metabolism, since formylglutamate is steps of purine synthesis. an intermediate in the conversion of the a-C atom of glycine into active formate. 2) Pyrimidine synthesis Urinary formate excretion rises in folic acid Folic acid is not concerned in the syn­ deficiency. Excretion is increased by oral thesis of the pyrimidine ring, but in the tryptophan but not histidine. introduction of the methyl group of thy-

ch2- nh2 ch2- nh I 5,10-CH ■> ' \ * HQ \ H4PteGlu \ +H,PleGlu FICUII 2, ,„tll,j„c. N H N H tion of carbon atom 8 I I into purine ring. -P Ribose-P

GAR FGAR BIOCHEMISTRY OF FOLIC ACID AND VITAMIN B]2 23 9

0 0 II II r HoN C— N * h2nTCvc 2 II ^ IO-CHO-H4PteGlu 8CH ------2------> 8CH + H4PteGlu / 0 = CH ^ C - N H2N i Ribose-P j Ribose-P

F i g u r e 3 . Introduc­ AICAR FAICAR tion of carbon atom 2 into purine ring. 0

CH

Ribose-P INOSINIC ACID mine (figure 4) and, in phage infected E. acid are impaired by the antagonists. The coli, the hydroxymethyl group of 5-hy- synthesis of thymidylate is believed to be droxymethylcytosine. The folate coenzyme the rate limiting step in DNA synthesis. is 5,10-methylenetetrahydrofolate. In the synthesis of thymidylate, CH2 is transferred Vitamin Bi2 from the folate coenzyme by thymidylate Vitamin Bi2, although found in most synthetase and simultaneously reduced to animal tissues, is almost exclusively syn­ CH3; concurrently the tetrahydrofolate thesized by micro-, either com­ initially formed is oxidized to dihydrofolate mensals in the animal’s digestive tract or which is then reduced to tetrahydrofolate ingested with animal food. The human is by and NADPH2. wholly dependent on ingested Bi2, being Recycling of the folic acid and the con­ tinuance of pyrimidine synthesis thus de­ unable to utilize any of the vitamin which pends upon the activity of dihydrofolate might be synthesized in the intestine. reductase. Folic acid antagonists which Vitamin Bi2 probably occurs in nature in inhibit the action of this enzyme inhibit its coenzyme form linked to a specific thymine synthesis; further, there is an ac­ protein. , the form of the cumulation of dihydrofolate and a decrease vitamin containing a group, does in the amount of tetrahydrofolate available not occur in the body, but is formed dur­ for conversion into other folate coenzymes. ing the extraction and purification pro­ Thus, other metabolic functions of folic cedures for obtaining the vitamin (active

OH

fl| : |l +5,IO-CHo-H4PteGlu> ^ ¡P0^ H2PieGlu F i g u r e 4. Introduc­ tion of methyl group into IS N °SV a pyrimidine. I d Rib o se-P d R ib o se-P

deoxyuridylic acid thymidylic acid 240 LUBRAN charcoal is used, which contains cyanide). to the nitrogen atom of the nucleotide and However, most assay procedures, whether to the cyanide group. Thirteen of the nine­ microbiological or radioisotopic, use cy- teen carbon atoms of the nucleus are anocobalamin as the reference material; the fully substituted with methyl groups or various forms of the vitamin are first con­ acetamide or propionamide residues. The verted to it during the preliminary extrac­ groups attached to the bridge carbon atoms tion procedures. are in the plane of the corrin nucleus; the other groups lie above or below the plane Chemistry of Vitamin Bn of the nucleus. Vitamin B12 has the structure shown in The nucleotide in vitamin B12 differs figure 5. It consists of two major portions: from the nucleic acid nucleotides in two a planar group closely, but not completely, respects: the base is neither a purine nor resembling the , and a nucleotide pyrimidine, but 5,6-dimethylbenziminazole; lying in a plane almost at right angles to the ribose linkage is «-glycosidic, not ¡3-gly- it. The - like structure is termed cosidic as in the nucleic acids. The ribose the corrin nucleus; unlike the porphyrins, is phosphorylated at C-3. The phosphate is there is a direct linkage between the two esterified with l-amino-2-propanol, which is a-carbon atoms of rings A and D. The cen­ combined through an amide link with the tral cobalt atom, which is trivalent and residue of ring D at C-17. positively charged, is linked to the four The third hydroxyl group of the phosphate nitrogen atoms of the reduced pyrrol rings, is ionized; the negative charge on the O atom and the positive charge on the Co

C0A/Hz atom make vitamin B12 an inner salt. The B12 structure without the cyanide group is termed a cobalamin. Other groups may take the place of cyanide. Im­ portant cobalamins are: , which contains an OH linked to the Co atom; aquocobalamin, which has H20 linked to the cobalt (it is the form in which hydroxocobalamin occurs in neutral or acid solution); sulphito, chloro, nitrito, bromo and thiocyanato groups may be attached to the Co by appropriate means. Coenzyme Bi2, finked to a specific pro­ tein, is the principal form in which B12 occurs in human and animal tissues. Two coenzymes are known: 5'-deoxyadenosylco- balamin in which adenosine (minus its OH at C 5') is linked at this atom to Co, and , in which a methyl group is directly attached to the cobalt atom. Both coenzymes are light sensitive in the presence of oxygen, yielding hydroxoco­ balamin. Cyano-, hydroxo- and other forms of Bi2 are rapidly converted in vivo into the coenzymes. BIOCHEMISTRY OF FOLIC ACID AND VITAMIN B 12 241

Biochemical Reactions of B 12 not in humans. The next two reactions are Coenzymes believed to occur in man, as well as in micro-organisms. These may be divided into two major groups: those requiring deoxyadenosylco- 5) L-methylmalonyl-CoA mutase reaction* balamin and those requiring methylco- This enzyme catalyzes the interconver­ balamin. As with folic acid, most of our sion of L-methylmalonyl-CoA (an inter- knowledge of the biochemistry of B12 has been obtained through the study of micro­ 0 Co in this enzyme refers to , not organisms. Those reactions known or be­ cobalt lieved to occur in the human will be dis­ cussed in some detail; the remaining I. Glutamate mutose reactions will be described briefly. CH-* I H HC—C-COOH Reactions requiring 5'-deoxyadenosylcobala- | nh2 min COOH In these reactions, the coenzyme-enzyme

complex combines with H from the sub­ 2. Dioldehydrase strate and transfers it to the adjacent C H 1- H atom. A group, originally attached to this H C -O fH HCH I + H 20 C atom, migrates to the C atom from which ( h/ c -I o H HC^O H ------the H has been detached. These changes result in an intramolecular rearrangement 3. Ethanolamine deaminase of the substrate. In some cases, water or H ____ , H HC-[NH^ HCH ammonia may be eliminated. In the case + NH, ( h/ c - o1h HC=0 of the ribonucleotide reductase reaction, w H L- which is more complex, the substrate is 4. j6-Lysine isomerase reduced in addition (figure 6). H H H H H II , H H H H H II 1) Glutamate mutase reaction HC-C^C-C-C-C-OH- -HC-C-C-C—C-C-OH LW iH | » H I H | H Glutamic acid is rearranged to form L- n h 2 NH2 n h 2 ihreo- ^-aspartic acid. 3 ,5 diaminohexanoic acid 2) Dioldehydrase reaction 5. L-M e)hylm alonyl-C oA mutase Ethylene or propylene glycol is con­ verted to acetaldehyde or propionaldehyde respectively.

3) Ethanolamine deaminase reaction COOH COOH Ethanolamine is converted to acetalde­ 6. Ribonucleotide reductase hyde.

Ir °H nH I H 4) ¡3-Lysine isomerase 8ase-C-C-C-C-C-0-P-P-(P)- H I I H H Lysine is converted to butyrate, acetate OH OH and ammonia by some clostridia. An inter­ mediate, /3-lysine, is converted to 3,5-di- I H H I H Base - C-C-C -C -C -O -P-P-(P) aminohexanoic acid. The further stages in H I H H OH the degradation are not clear. F ig u r e 6 . Reactions requiring These four reactions occur in bacteria, deoxyadenosylcobalamin. 242 LUBRAN mediate in the enzymatic conversion of Study of this reaction in bacteria has propionate to succinate) and succinyl-CoA. shown that two different reductases exist; The reaction involves the transfer of the one is B12 dependent, the other is indepen­ H from C-3 of the substrate to C-5' of the dent of Bi2. Mammalian ribonucleotide deoxyadenosyl moiety of the coenzyme. The reductase requires Mg++ and acts on CoA- carboxyl group migrates from ribonucleotide disphosphate. It is not C-2 to C-3 of the substrate and the H is stimulated by Bi2 coenzyme and is prob­ transferred from the coenzyme to C-2. ably not B12 dependent. Bi2-depleted bone There is decreased activity of this enzyme marrow, obtained from patients with system in vitamin Bi2 deficiency; urinary pernicious anemia, showed no stimulation excretion of is in­ by Bi2 or the deoxyadenosyl coenzyme.5 creased from the normal value of less than Thus, there is no evidence to show that 4 mg in 24 hours to over 6 mg methyl­ ribonucleotide reductase activity in man is excretion in normals is un­ dependent on deoxyadenosylcobalamin. affected by an oral dose of 10 gm of valine; this dose causes an increased excretion in Reactions Requiring Methylcobalamin patients with pernicious anemia. Increased excretion of methylmalonic acid is not con­ These reactions involve transfer of a sistently found in patients with other forms methyl group from 5-methyltetrahydro- of B12 deficiency. It is normal in patients folate to an appropriate substrate. Methyl­ with folic acid deficiency who do not have cobalamin, bound to a reducing enzyme, concurrent B12 deficiency (these two de­ acts as intermediate transfer agent in the ficiencies often occur together). Normal reaction. Only the first reaction described methylmalonic acid excretion is restored in below is known to occur in man. patients with B]2 deficiency who are treated with parenteral Bi2. Folic acid administra­ 1) Methionine synthesis tion in these patients is without effect. In this reaction, methionine is formed These clinical observations point strongly to from by transfer of a methyl the existence of methylmalonyl-CoA mutase group from 5-methyltetrahydrofolate; in the human. FADH2, S-adenosylmethionine and methyl­ 6 ) Ribonucleotide reductase reaction cobalamin are required. The de novo syn­ thesis of the methyl group takes place as This enzyme catalyzes the reduction of described earlier through the formation of ribonucleotides to deoxyribonucleotides, 5-methyltetrahydrofolate. Methionine syn­ which are required for DNA synthesis. The thesis serves as the means of renewing the substrate is a ribonucleotide diphosphate tetrahydrofolate. or triphosphate. The reaction involves the substitution of an OH group by H in the SH.OH2.CH2.CHNH.COOH +5-CH ,-H 4 PteGlu -* homocysteine 2' position of the ribosyl moiety by a com­ CH,S.CH2.CH,.CHNH o.COOH + H 4 PteGlu methionine plicated mechanism involving transfer of H from a group of a low molecular The reaction mechanism is complex. Ini­ weight protein to the C-5' of the coenzyme- tially, the reducing enzyme is combined adenosyl moiety. Here it is exchanged for with a cobalamin. In the presence of the an OH. This is not an intramolecular re­ other factors, the Co is reduced to the arrangement, as occurs in the other reac­ monovalent state, in which it readily ac­ tions described, but transfer of H, via the cepts the CH3 group from the S-adenosyl­ coenzyme, from a hydrogen donor. methionine, forming enzyme-bound methyl- BIOCHEMISTRY OF FOLIC ACID AND VITAMIN B12 243 cobalamin. The methyl group is transferred Of the many biochemical reactions in­ from this as a carbonium ion to homo­ volving B12, only two are believed to occur and the cobalamin is remethylated in man: the isomerisation of L-methyl- by a methyl group donated by the 5-methyl- malonyl-CoA to succinyl-CoA (not affected tetrahydrofolate. In the reaction, methyl by folate; disturbance of this enzyme action groups are donated by 5-methyltetrahydro- is postulated as the cause of the degenera­ folate; S-adenosylmethionine acts as a tive disease of the nervous system due to primer (i.e., it initiates the reaction) and B12 deficiency), and the of methylcobalamin is the transfer agent. A homocysteine to methionine, which is folate Bi2-independent pathway for methionine dependent. It is this latter reaction which synthesis exists in some bacteria. It is of forms the common ground between folate fundamental importance to determine and vitamin Bi2. The folate coenzymes are whether methionine synthesis can occur interconvertible, with the exception of 5- independently of B]2 in the human. This methyltetrahydrofolate. The probable way question is discussed in the next section. in which tetrahydrofolate is regenerated is through the synthesis of methionine. 2) Methane formation The “methyltetrahydrofolate trap” hy­ Methanol is converted to methane by pothesis postulates that, in fact, 5-methylte- bacteria in sewage sludge. Methylcobalamin trahydrofolate can be converted to other is involved. The mechanism is similar to folate derivatives only by the cobalamin- that described for methionine synthesis. dependent methyltransferase reaction; this reaction is seriously diminished in Bi2 de­ 3) Actetate synthesis ficiency. The result is that the total body Both 5-methyltetrahydrofolate and BJ2 pool of folate is largely trapped in the are involved in the total synthesis of form of methyltetrahydrofolate and reac­ acetate from C 02. As in the two reactions tions depending on the other folate co­ described above, a protein-bound methyl­ enzymes are diminished. In particular, cobalamin complex is formed as an inter­ purine and pyrimidine are mediate. diminished and therefore DNA production is impaired. Megaloblastosis results. The Interrelationships of Vitamin Bi2 and hypothesis depends upon the demonstra­ Folic Acid tion that the cobalamin-independent path­ way, known to exist in some bacteria, does Folic acid deficiency and pernicious not exist in man, or is of little significance. anemia produce the same cellular changes This has not, as yet, been satisfactorily in the human bone marrow. Tissue cul­ determined by direct methods. ture studies of normal human marrow and Some indirect evidence supports the hy­ megaloblastic marrow, involving incorpora­ pothesis. Methyltetrahydrofolate is the tion of tritium-labelled precursors into principal monoglutamate coenzyme of DNA and RNA of megaloblasts and plasma and liver. In Bi2 deficiency asso­ erythroblasts, suggest that, in these defici­ ciated with a normal intake of folate and ency states, there is a failure to complete methionine, there is often an increase in DNA synthesis prior to mitosis, i.e. there is plasma methyltetrahydrofolate; this is con­ prolongation of the S and G-2 phases of sistent with the hypothesis. The crucial the cell cycle. These observations suggest test of the methyltetrahydrofolate trap a close association between B12 and folate hypothesis would be the demonstration of during hematopoiesis. tissue deficiency of folate derivatives, other 244 LUBRAN than methyltetrahydrofolate, in patients the clinically documented relationship be­ with pernicious anemia in relapse, on ade­ tween vitamin Bi2 and folic acid in cell quate dietary folate and methionine.3 Di­ metabolism. rect measurements on marrow and liver have not been performed. Current methods References have inadequate sensitivity. 1. C h a n a r in , I.: The Megaloblastic Anaemias: However, indirect evidence of folate F. A. Davis Co., Philadelphia, 1969. enzyme levels in tissues can be obtained 2. H u e n n e k e n s , F. M.: Folic acid coenzymes in by studying in these patients enzyme reac­ the biosynthesis of purines and pyrimidines. tions which are folate dependent. The in­ Vitamins Hormones 26:375-394, 1968. 3. N ix o n , P. J. a n d B e r t in o , J. R.: Interrelation­ creased excretion of FIGlu and amino- ships of vitamin Bi2 and folate in man. Amer. imidazolecarboxamide in the urine of Bi2 J. Med. 48:555-561, 1970. deficient patients, diminished by treatment 4. S il b e r , R. a n d M o l d o w , C. F.: The bio­ with folate, is in keeping with the hypoth­ chemistry of Bi2-mediated reactions in man. Amer. J. Med. 48:549-554, 1970. esis. In vitro experiments on the thymid- 5. S il b e r , R., F u j io k a , S., M o l d o w , C. F ., a n d ylate synthetic activity of cells from bone Cox, R.: Altered regulation of deoxyribonu- marrow aspirates suggest that there is a cleotide synthesis in B12 or . deficiency of methylenetetrahydrofolate in Clin. Res. 18:416, 1970. 6. S m i t h , E. L.: Vitamin Bi2 J. W iley and Sons, the marrow cells of cobalamin-deficient as Inc., New York, 1965. well as folate deficient patients. Although 7. S t o k s a d , E. L. R. a n d K o c h , J.: Folic acid there are some contradictory experiments, metabolism. Physiol. Rev. 47:83-116, 1967. the methyltetrahydrofolate trap hypothesis 8. W e is s b a c h , H. a n d T a y l o r , R. T .: Metabolic role of vitamin Bi2. Vitamins Hormones 26: provides at present the best explanation for 385-412, 1968.

SPRING MEETING of the ASSOCIATION OF CLINICAL SCIENTISTS

Topics Advances in Clinical Science Elkhart, Indiana April 27-30, 1972