Folic acid metabolism in patients with malignant hematologic diseases has been studied by assaying the urinaryexcretion of folic acid following p.o. administration of atest dose (15) or the rate of plasma clearance of an i.v. doseof folic acid (8). The decreased excretion or rapid clearance rate noted in many patients with advanced diseasewas not correlated with the severity of the disease or withthe responsiveness to therapy, and definite conclusions relative to the state of the disease could not be drawn. Careyet al. (7) have recently reported elevated formiminoglutamate levels in a significant number of patients with malignancies ; this suggests that folate deficiency may be fairlycommon in such patients. However, formiminoglutamateis a measure of many phenomena unrelated to folate deficiency, such as liver disease (22). The serum folate levelas measured by Lactobacillus casei proved to be a sensitiveindex of folate status in man (3, 17). Many workers haveconfirmed this (9, 16, 20, 26).

We have determined the levels of serum folate and serumvitamin B12 in a large number of patients with malignanthematologic diseases and have attempted to correlate theresults with the status and responsiveness of the disease.The results of that study form the basis of this report.

MATERIALS AND METHODS

Blood was obtained from 225 fasting, previously untreated patients in sterile, cotton-stoppered test tubes andallowed to clot for 3 hr at room temperature. The clotwas then rimmed, the tube was centrifuged for 15 mm, andthe supernatant serum was aspirated and frozen until assay was made, which in all instances was within 1 month.The sera were tested without the diagnosis and the status

1 This study was aided in part by USPHS Grants CA-04457,

AM-09062,and AM-04971and in part by the Albert A. List, Frederick Macklin, and Anna Ruth Lowenberg Funds.

Received for publication January 19, 1965; revised July 19,1965.

of disease being revealed to the persons performing theassay. Patients receiving vitamin pills were excludedfrom the study. Serum folate levels and vitamin B12 activity were determined microbiologically with the use ofLactobacillus casei (3) and Euglena gracilis (24), respectively, as the assay organisms. The normal values in ourlaboratories range between 7 and 25 ng2 per ml for folate.Only values below 5 ng/ml are regarded as being significantly low. The normal values for the serum B12 activityrange between 200 and 900 pg2 per ml. In our laboratoriesthe S.D. of individual assays and the day-to-day variationsin a control individual are within 10 %. Twenty patients

with nonmalignant diseases were tested as controls. Ofthese, 12 were recovering from a myocardial infarction, 4had rheumatoid arthritis, 2 had pulmonary emphysema,1 had a duodenal ulcer, and 1 was admitted for removal ofa benign polyp of the colon.

Some patients were receiving antibiotic therapy ; however, this does not influence the serum folate levels. Thelevel of any antibiotic in the serum dilutions used in thetests is so low that no effect on the growth of the nucroorganisms is seen.3 In addition, the sera are autoclavedin preparation for the assay. The broad-spectrum anti

biotics are heat labile and are thus destroyed in the process.Sulfonamides could possibly inhibit growth of L. casei;however, the addition of p-aminobenzoic acid to the basalmedium obviates this effect.

CRITERIA FOR STATUS OF DISEASE

Acute leukemia.—The status of disease was divided into3 categories : (a) acute or active, (b) partial remission, and(c) complete remission, as determined by the criteria of the

Clinical Studies Panel of the Cancer Chemotherapy National Service Center (6).

2 The designations for millimicrogram and micromicrogram areng (nanogram) and pg (picogram), respectively.

3 H. Baker, unpublished data.

1933

Serum Folate and Serum Vitamin B12in Patients withMalignant Hematologic Diseases1

BARTH HOOGSTRATEN, HERMAN BAKER, AND HARRIET S. GILBERT

Departments of Hematology and Chemistry, The Mount Sinai Hospital, New York, New York

SUMMARY

The serum folate and serum vitamin B@ levels were determined in a large numberof patients with malignant hemotologic diseases. Low values of folate were noted

mostly in patients with active disease, whereas the majority of cases in complete remission or with mild disease had normal values. No such correlation between thestatus of disease and serum vitamin B12 levels was found. In chronic myelocyticleukemia the number of white blood cells is inversely proportional to the folate level.Serum folate determinations may prove helpful in predicting response to daily p.o.antifol therapy in most patients.

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1934 Cancer Research

Chranic leuke@nia.—The categories for this disease are:(a) active, with an organ enlargement extending below theumbilicus, a WBC above 100,000/cu mm or hemoglobinbelow 10 gm% , and almost complete inability to work;(b)partialremission,withanorganenlargementnotcxtending below the umbilicus, a WBC between 25,000 and100,000/cu mm, hemoglobin between 10 and 12 gm %, anda performance status of at least 50 % ; @c)mild disease,with the virtual absence of all evidence of disease and anormal performance status. The “blastic phase― ofchronic myelocytic leukemia was defined by marked anemia, thrombocytopenia, and a majority of blasts in theperipheral blood and bone marrow.

Lyinphoma.—A modified Craver's classification was used(10) as follows : (a) active, in which generalized disease isaccompanied by constitutional signs, hemoglobin below 10gm% , and performance status below 50 % ; (b) partial re

mission, in which the disease is in more than one area andthere are some constitutional signs, anemia with hemoglobin above 10 gm % , and a performance status of at least50 % of normal ; (c) complete remission, in which there are“unicentric―disease without constitutional signs, no anemia, and normal performance status.

Multiple inyelorna.—The 3 categories were defined asfollows : (a) active, in which there were anemia with hemoglobin below 10 gm% and complete inability to work; (b)moderate disease or partial remission, in which there wereanemia with hemoglobin between 10 and 12 gm% and aperformance status of at least 50 % ; (c) mild disease orcomplete remission, in which there were no anemia and anormal performance status. All patients with multiplemyeloma had diagnostic bone marrow findings. Caseswith solitary plasmacytomas were not included.

RESULTS

In Charts 1 and 2 the serum folate and vitamin B12levelsof each patient when seen initially are given and categorized by disease and status of disease.

Acute myeloblastic leukemia (AML)4.—Assays for folatelevels were performed on the sera of 42 patients, with 35sera assayed for vitamin B12. When first seen, patientswere rarely in complete remission ; 3 patients with completeremission after anti-leukemic therapy will be discussedlater. Only in the active disease were very low folatelevels noted ( < 3.0) ; 6 cases with active disease had normal or high levels. Seven of 12 patients in partial remission had a low level of folate. The difference between the

levels in active disease and in partial remission has a lowdegree of significance with P > 0.15 (Table 1).

The serum B12 level was high in 17 patients, normal in17, and slightly diminished in 1 case.

Chronic myelocytic leukemia (CML).—Thirty-six serawere assayed for the folate level, and the serum vitaminB12 level was determined in 33. All patients in completeremission had normal levels, whereas 2 of 7 cases with apartial remission had low values. Only 2 normal values

4 The following abbreviations are used: AML, acute myeloblas

tic leukemia; CML, chronic myelocytic leukemia; ALL, acutelymphoblastic leukemia; CLL, chronic lymphocytic leukemia;Ly. Sarc., lymphosarcoma; Leukosarc., leukosarcoma; C.R., cornplete remission; P.R., partial remission; and MCV, mean corpuscular volume.

SERUMFOLATE(ng/mi )

â€â€”ACTIVE

0 - PARTIAL REMISSION.- COMPLETE REMISSION

a—BLASTIC PHASE OF CML

CHART 1.—Initial serum folate levels grouped according todisease and disease status. Abbreviations used are: AML,acute myeloblastic leukemia; CML, chronic myelocytic leukemia;ALL, acute lymphoblastic leukemia; CLL, chronic lymphocyticleukemia ; Ly. Sarc ., lymphosarcoma.

@45OO

I

@0:

a

C L L Hodgkin's Ly. sate. MyslomaAMLCMLALL

x —ACTIVE0—PARTIAL REMISSION

5— COMPLETE REMISSIONs—BLASTIC PHASE OF CML

CHART 2.—Initial serum vitamin B12 levels grouped accordingto disease and disease status. Abbreviations used are: AML,acute myeloblastic leukemia; CML, chronic myelocytic leukemia;ALL, acute lymphoblastic leukemia; CLL, chronic lymphocyticleukemia ; Ly. Sarc. , lymphosarcoma.

were obtained in the group with active disease; 9 patientswere in a “blasticphase,― and the lowest levels were obtamed in this group. The average folate level of the 10patients with active disease was 5.5 ng/mi, whereas forthe 9 cases with a blastic phase it was 2.8. On the @%level of significance the difference between active diseaseand remission (partial plus complete) is marked,P < 0.0001. When the patients in a blastic phase were

Vol. 25, December 1965

SERUMB 12

(pg/mi5000

2000

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PartialDisease

categoryActive @BlasticActivemissionAML―

(42cases)Mean5.9

8.3Difference2.4“t―1.33P>0.15CML

(36cases)Mean43b

10.610.6 11.92.85.5Difference6.31.32.7“t―5.000.701.56P<0.0001>0.40>0.10CLL

(25cases)Mean8.5

14.716.910.7Difference6.2—6.2“t―1.95—1.41P>0.05>0.10Hodgkin's(26

cases)Mean3.27.76.310.3Difference4.54.3“t―1.732.12P<0.05<0.05Ly.

Sarc.(37cases)Mean6.6

11.48.214.5Difference4.86.3“t―3.082.68P<0.002<0.01Mult.

Myel.(39cases)Mean6.1

11.09.315.5Difference4.96.2“t―3.832.95P<0.0001<0.01

HOOGSTRATEN et al.—Serum Folate and Vitamin B12 in Hematologic Malignancies 1935

TABLE 1COMPARISON OF MEAN FoLic ACID LEVELS (ng/ml)

U)UiU)‘I0U.0

UI

z

-3 3-4 4-5 5-6 6-7 >7SERUM FOLATE ( rig /mi)

CHART 3.—Serum folate levels of 225 patients grouped according to status of disease.

this group had low levels ; none had levels below 3 ng/ml.The average folate level in the active disease category wasnormal, although it was lower than that of the remissioncategories—8.5 versus 14.7 ng/ml.

Hodgkin's disease.—In this group of 26 patients, the 6cases in C.R. had normal or borderline low values. All 8patients with active disease had low or borderline lowvalues. Seven patients in P.R. had normal or near normal levels ; 5 had low levels. These differences are significant : P < 0.05 for active disease and remissions andP < 0.05 for the difference in partial or complete remission. The vitamiii B12 level in 18 of the 19 cases studied

was normal.Lymphosarcorna.—Thirty-seven patients had a folate

assay, and 10 of 21 cases with active disease had a normalor borderline low level, whereas the remaining 11 had lowvalues. The 8 patients in P.R. had normal or borderlinelow levels. Only 1 subject in C.R. had a low level.Again there are significant comparisons between the levelsin active disease and in remission, P < 0.002, and in partial remission or complete remission, P < 0.01. The vitamin B12 content of the sera of 21 patients was normal ; itwas low in 1 case.

Multiple myeloma.—Assays for folate levels were performed in 39 patients. In this disease a comparison of thelevels in active disease and remission, or in P.R. and C.R.,shows the greatest difference: P < 0.0001 and P < 0.01,respectively. The serum vitamin B12 of 32 cases was normal or elevated ; it was low in 2 cases.

In Chart 3 the folate values of all patients are groupedaccording to status of disease, for values below and above7 ng/ml, and compared to the folate values of 20 patientswith nonmalignant diseases. Low levels were usuallyfound in active disease and in P.R., whereas the majorityof the cases in C.R. or with mild disease had normal levels.Only 2 of the cases without a malignancy had a borderlinelow value, and both had chronic blood loss.

The effect of various chemotherapeutic agents on the

a AML, acute myeloblastic leukemia; CML, chronic myelo

cytic leukemia; CLL, chronic lymphocytic leukemia; Ly. Sarc.,lymphosarcoma; Mult. Myel., multiple myeloma.

b Includes blastic phase.

excluded, the difference, though less marked, was stillsignificant, P < 0.05. The WBC is inversely proportionalto the serum folate level. The average folate value forpatients with less than 25,000 WBC was 11.9; with a WBCbetween 25,000 and 100,000 it was 7.4, and with a WBCover 100,000 it was 4.2. No significant differences werenoted for the vitamin B12 levels, although as a group theaverage for patients in remission was lower than that forpatients with active disease. For C.R., P.R., active disease, and blastic phase, the averages were respectively,2175, 2165, 3300, and 3970 pg/mI.

Acute lymphoblastü@leukemia (ALL).—Of the 20 patientswith active disease, 9 had a low folate level, 5 had a borderline level, and 6 had a normal level. A normal level wasfound in 1 patient with a C.R. The serum B12 level wasnormal in 19 patients and low in 1.

Chronic lymphatic leukemia (CLL).—Few patients iii

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DiagnosisSubjectDateSFAA (ng/ml)Status ofdiseaseRemarksAML―A.B.12/14/59

1/21/603/11/604/13/60<1.0

2.73.76.4Active

ActiveP.R.CR.64,000

WBC700 WBC

1,250WBC3,600WBCAMLM.

M.11/1/582/27/595/13/59

8/4/59<1

.04.610.8

9.4Active

P.R.C.R.P.R.MegaloblastosisEarlyrelapseAMLA.

Z.11/4/597/7/60

10/4/6012/6/608.6

16.210.33.8C.R.

C.R.ActiveActiveEarly

relapseAMLN.

S.7/19/628/7/625.5 10.4Active C.R.80,000

WBC3,100WBCCMLA.B.3/18/60

5/11/606.7 2.2P.R.Blastic49,000WBCCMLK.3/24/60

5/11/607.2 1.7P.R. BlasticMC\7106ALL

Leukosarc.W.

D.

J. G.11/1/6111/29/61

3/25/614/4/61

4/12/614/20/612.2

9.93 .62 .84.16.0Active

C.R.ActiveActiveActiveActiveAfter

6-MP180,000 WBC181,000 WBC74,000 WBC

1,700WBCHodgkin'sR.E.3/10/60

10/22/607/10/615.7

11 .65.8P.R.

CR.P.R.After

HN2Hodgkin'sD.

S.12/23/5912/30/597/3/60

10/11/607.5

7.82.71.1P.R.

P.R..Active

ActiveLy.Sarc.H. B.5/16/60

7/10/6011/29/615.3

11.915.1P.R.

C.R.C.R.After

uracilmustardLy.

Sarc.D. K.5/23/607/13/604.3 9.9Active P.R.After HN2

1936 Cancer Research Vol. 25, December 1965

TABLE 2CHANGES OF SERUM FOLATE DURING DIFFERENT PHASES OF DISEASE

a AML, acute myeloblastic leukemia; CML, chronic myelocytic leukemia; ALL, acute lymphoblas

tic leukemia; Leukosarc., leukosarcoma; Ly. Sarc., lymphosarcoma; P.R., partial remission; C.R.,complete remission; HN2, nitrogen mustard; MCV, mean corpuscular volume; 6-MP, 6-mercaptopurine.

gm % , MCV 114, and a megaloblastic marrow. Theserum B12 absorption studies were normal. Two monthslater a repeat marrow showed myeloblastic leukemia andmegaloblastosis. With anti-leukemic therapy, 1st a partial and later a complete remission was obtained. Themarrow became normoblastic without adding folic acid tothe treatment regime. In P. R., the folate level was 4.6ng/ml; it subsequently rose to 10.8 ng/ml in C.R. The3rd case was a 42-year-old woman with Hodgkin's diseaseof 2.5 years' duration. On her last admission shortly before death from severe pulmonary involvement, a megaloblastic bone marrow was obtained. The serum vitaminB12 level was 885 pg/mi, whereas the folate level was 0.9ng/ml (she had received no vitamin therapy).

DISCUSSION

There are several possible causes of the low serum folatelevels observed in this study : (a) The 1st is decreased dietary folate level because of inanition. This possibility wasstudied by Rama Rao et al. (23) in a large group of patients

assays of the serum vitamin levels was determined. Noeffect was noticed from the alkylating agents and 6-mercaptopurine ; however, methotrexate therapy resulted ininhibition of growth of the microorganisms, which lastedfor 3—4days after discontinuation of therapy and necessitated a delay of at least 1 week before repeat assays couldbe made. Significant changes of the folate levels duringdifferent phases of the disease were noted (Table 2). Theywere especially noteworthy in the 1st 2 cases, A. B. andM. M., in whom, while in remission, normal folate valueswere obtained, which had risen from previously extremelylow levels.

Macrocytic anemia was seen in 3 cases. The 1st, a 56-year-old man with a blastic phase of chronic myelocyticleukemia, had a serum B12 level of 3100 pg/ml and a folatelevel of 1.4 ng/ml associated with a severe macrocyticanemia and many hypersegmented polynuclears. Unfortunately, several bone marrow aspirations failed to provide marrow for study. The 2nd patient was a 57-yearold female (M. M., Table 2) with a hemoglobin of 4.5

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HOOGSTRATEN et al.—Serum Folate and Vitamin B12 in Hematologic Malignancies 1937

with various malignant tumors (excluding leukemia andlymphoma). The mean folate levels for patients in a good,and in a poor, general condition were almost identical. Ittherefore appears as if the nutritional status does not ap-.preciably affect the folate level, unless there is markedmalnutrition. This was not the case in our patients. (b)The 2nd possible cause is poor absorption of dietary folicacid. This aspect was studied by Chanarin et al. (8) andin some of our patients. Oral test doses of folic acid causeda sharp rise of the serum folate level in all but 1 patient.This patient showed other evidence for malabsorption andwas excluded from the study. (c) The 3rd possible causeis poor deconjugation of dietary folate. In 8 of our patients thepostprandialserum folate levelwas higher than thefasting level. However, this aspect should be studied inmore cases. (d) The 4th possible cause is increased demand for folate by neoplastic cells. Although this p05-sibility has been postulated for many years, at present itcan be proven only through exclusion of all other possiblecauses. It is a likely explanation for a low folate level inthose patients in whom normal absorption of folic acid hasbeen demonstrated and in whom, after adequate treatmentwith folate, a rapid plasma clearance rate is present.

Correlation between the serum folate level and responseto anti-folate therapy has previously been reported (18).This correlation was again shown in 12 patients in whomdaily p.o. anti-folate therapy resulted in remissions onlyin those cases with a normal folate level. However, whena low serum folate level was found during the subsequentrelapse, a 2nd remission with antifols could not be obtamed. Parenteral antifol therapy given intermittentlyill large doses results in a greater number of remissions.

The blood antifol level is temporarily very high, and thusit is conceivable that even leukemic cells with a high folicacid reductase content (12) or cells with decreased uptakeof antifol (13, 19) are affected.

Increased folate activity in the leukocytes of patientswith Ai'vIL was found by Swendseid et al. (25), and a normal level of citrovorum factor was reported in the leukocytes of 7 patients in C.R. by Ellison and Hutchison (1 1).Bachi and Coscia (1) reported a rise of folic acid excretionafter a p.o. test dose in patients with clinical improvementafter therapy. Similarly, we noted low folate levels inmost patients with active disease and a return to normallevels when a remission was obtained. Four patients withactive disease had normal folate levels. It is possible thatthe disease has to be active for some time before depletionof the folate stores takes place. An indication of this canbe seen in patient A. Z. (Table 1). When a relapse was 1stnoted after a C.R., the folate level was within normallimits ; 2 months later, while the patient was still in relapse,a low level was obtained.

Bachi et al. (2) studied the urinary excretion of folic acidin 17 patients with Hodgkin's disease, and Girdwood (15)

reported 6 cases ; a low excretion was more frequent in patients with disseminated disease. In our study a distinctdifference was noted in the serum folate level of patientsin C.R. and of those with P.R. or active disease. Similarfindings were seen in patients with lymphosarcoma andmultiple myeloma.

Unlike the values for folate, marked differences duringthe various stages of disease were not seen in the serum

B12 levels ; only 7 of 171 patients had a decreased level ofB12. High values were seen in AML and CML. Beardet al. (4, 5) reported a mean serum B12 level of about 3times normal in AML. The mean value in the cases reported here and in 14 patients previously studied was 1050pg/ml with a range of 140—4300. High values in Ci@vILhave been reported (4, 14) and were again noted in ourstudy. Normal levels were seen in ALL, CLL, Hodgkin'sdisease, lymphosarcoma, and multiple myeloma. In multiple myeloma an average serum vitamin B12 level of 270pg/mi, half that of normal subjects, was reported byMandema et al. (21) in 13 patients. In our 34 cases therange was from 105 to 3300 pg/mi, with an average levelof 515 pg/mi.

ACKNOWLEDGMENT

The authors are indebted to I)r. P. G. Reizenstein, formerlyof the Brookhaven National Laboratories, for his contributionsto this study.

REFERENCES

1. Bachi, C., and Coscia, G. Contributo allo Studio del Metabolismo dell'Acido Folico nelle Leticemie. VII. Congr.Intern. Soc. Hematology, pp. 150—53. New York: Grime &Stratton, Inc., 1958.

2. Bachi, C., Coscia, G., and Rossi, M. Studio sEll Metabolismodell'Acido Folico nd Morbo di'Hodgkin e nelle NeoplasieMaligne. Minerva Med., 50: 2349—Si,1959.

3. Baker, H., Herbert, V., Frank, 0., Pasher, I., Hutner, S. H.,Wasserman, L. R., and Sobotka, H. A Microbiologic Methodfor Detecting Folic Acid Deficiency in Man. Clin. Chem.,5: 275—80,1959.

4. Beard, M. F., Pitney, W. R., and Sanneman, E. H. SerumConcentrations of Vitamin B12 in Patients Suffering fromLeukemia. Blood, 9: 789—94,1954.

5. Beard, i@1.F., Pitney, W. R., Sanneman, E. H., Sakol, M. J.,and Moorhead, IT. H. Serum Concentrations of VitaminB,2 in Acute Leukemia. Ann. Internal Med., 4!: 323—27,1954.

6. Bisel, H. F. Letter to the Editor. Blood, 11: 676—77,1956.7. Carey, R. W., Brena, G. P., and Drant, M. J. Urinary Formi

minoglutamic Acid Excretion in Patients with NeoplasticDisease.Cancer,17:713—22,1964.

8. Chanarin, I., Mollin, D. L., and Anderson, B. B. FolicAcid Deficiency and Megaloblastic Anaemias. Proc. Roy.Soc. Med., 51: 757—63,1958.

9. Cooper, B. A., and Lowenstein, L. Evaluation of Assessmentof Folic Acid I)eficiency by Serum Folic Acid Activity Measured with L. casei. Canad. Med. Assoc. J., 85: 987—88,1961.

10. Craver, L. F. The Nitrogen Mustards: Clinical Use. Radiology, 50: 486—93,1948.

11. Ellison, R. H., and Hutchison, D. J. Metabolism of FolicAcid and Citrovorum Factor in Leukemic Cells. In: J. W.Rebuck, F. H. Bethell, and H. W. Monto (eds.), The Leukemiss: Etiology, Pathophysiology and Treatment, pp. 467—77.New York: Academic Press, Inc., 1957.

12. Fischer, G. A. Increased Levels of Folic Reductase as aMechanism of Resistance to Amethopterin in LeukemicCells. Biochem. Pharmacol., 7: 75—80,1961.

13. . Defective Transport of Amethopterin (Methotrexate) as a Mechanism of Resistance to the Antimetabolitein L5178Y Leukemic Cells. Ibid., 11: 1233—34,1962.

14. Ghosh, S., Banerji, D. K., and Chatterjea, J. B. VitaminB,, in Leukemia. Bull. Calcutta School Trop. Med., 6: 2-4,1958.

15. Girdwood, R. H. Folic Acid Excretion Studies in the Investigation of Malignant Disease. Brit. Med. J., 2: 741-45, 1953.

16. Hansen, H. A., and Weinfeld, A. Metabolic Effects and Diagnostic Value of Small Doses of Folic Acid and B,2 in Megaloblastic Anemias. Acta Med. Scand., 172: 427-43, 1962.

17. Herbert, V., Baker, H., Frank, 0., Pasher, I., Sobotka, H.,

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1938 Cancer Research

and Wasserman, L. R . The Measurement of Folic Acid Activity in Serum: A Diagnostic Aid in the Differentiation ofthe Megaloblastic Anemias. Blood, 15: 228—35,1960.

18. Hoogstraten, B., Baker, H., and Reizenstein, P. G. Correlation between Folic Acid Activity and Response to Anti-folateTherapy. Ibid.,18:787,1961.

19. Jukes, T. H., and Broquist, H. P. Sulfonamides and FolicAcid Antagonists. In: R. M. Hochster and J. H. Quastel(eds.), Metabolic Inhibitors, pp. 481—534. New York: Academic Press, Inc., 1963.

20. Lindenbaum, J., and Klipstein, F. A. Folic Acid Deficiencyin Sickle Cell Anemia. New Engl. J. Med., 269: 875—82,1963.

21. Mandema, E., Faber, J. G., de Vries, J. A., and Nieweg, H. 0.Gehalte aan Vitamine B,, in het Serum van Lijders aan Multipel Myeloom. Ned. Tijdschr. Geneesk., 100: 3588—89,1956.

22. Merritt, A. D., Rucknagel, D. L., Silverman, M., and Gardi

ncr, R. C. Urinary Urocanic Acid in Man: The Identification of Urocanic Acid and the Comparative Excretions ofUrocanic Acid and N-Formiminoglutamic Acid after OralHistidine in Patients with Liver Disease. J. Clin. Invest.,41: 1472—83,1962.

23. Rama Rao, P. B., Lagerlof, B., Einhorn, J., and Reizenstein,P. G. Folic Acid Activity in Leukemia and Cancer. Cancer Res., 25: 221—24,1965.

24. Ross, G. I. M. Vitamin B12 Assay in Body Fluids using Euglena gracilis. J. Clin. Pathol., 5: 250—56,1952.

25. Swendseid, M. E., Bethell, F. H., and Bird, 0. D. The Concentration of Folic Acid in Leukocytes. Observation on Normal Subjects and Persons with Leukemia. Cancer Res., 11:864—67,1951.

26. Waters, A. H., and Mollin, D. L. Studies on the Folic AcidActivity of Human Serum. J. Clin. Pathol., 14: 335-44, 1961

Vol. 25, December 1965

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1965;25:1933-1938. Cancer Res   Barth Hoogstraten, Herman Baker and Harriet S. Gilbert  Hematologic Diseases

in Patients with Malignant12Serum Folate and Serum Vitamin B

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