J. clin. Path. (1960), 13, 440.

FOLIC ACID AND OTHER ABSORPTION TESTSIN LYMPHOSARCOMA, CHRONIC LYMPHOCYTICLEUKAEMIA, AND SOME RELATED CONDITIONS

BY

W. R. PITNEY, R. A. JOSKE,* AND NORMA L. MACKINNONFrom the Department of Haematology, Royal Perth Hospital, and the Department of Medicine, University

of Western A ustralia, Perth, Western A ustralia

(RECEIVED FOR PUBLICATION MAY 11, 1960)

An improved folic acid absorption test is described in detail.Using the folic acid and other absorption tests, a study of intestinal function has been made

in eight patients with lymphosarcoma, eight with chronic lymphocytic leukaemia, two withHodgkin's disease, and one each with reticulum cell sarcoma, Whipple's disease, and macro-globulinaemia. It was found that laboratory evidence of intestinal malabsorption was extremelycommon in patients with this group of diseases.The folic acid absorption test was the most sensitive single index of malabsorption of the tests

used in this study.Correlation between the different absorption tests was poor, and there does not appear to be

a characteristic biochemical profile of intestinal function in these patients.

The pathology and clinical features of primarylymphosarcoma and allied tumours of thealimentary tract have been described by severalauthors, including Spellberg and Zivin (1949) andSkrimshire (1955). These tumours are uncommon,but may produce dyspepsia, gastrointestinalhaemorrhage, or intestinal obstruction. A rarecomplication is the development of steatorrhoeaand overt malabsorption. This was firstdescribed by Fairley and Mackie (1937), and therelevant literature has recently been reviewed bySleisenger, Almy, and Barr (1953), who reportedfour further cases.

In the majority of patients with lymphosarcoma,involvement of the gastrointestinal tract andmesenteric lymph nodes is an incidental patho-logical finding associated with infiltration of manyother tissues. It rarely produces significantsymptoms, and is not regarded as important in thenatural history of the disease. In a recent patient(As) who underwent splenectomy for acquiredhaemolytic anaemia associated with lympho-sarcoma, we were impressed by th2 appearance ofstiffened oedematous bowel at operation, althoughthe patient had noted only slight symptoms ofintestinal dysfunction. We therefore decided toinvestigate intestinal function in an unselected*Adolph Basser Research Fellow in Medicine, Royal Australasian

College of Physicians.

series of patients with lymphosarcoma not arisingprimarily in the alimentary tract. Since somewhatsimilar infiltration of the intestinal wall andmesenteric lymph nodes may also occur in patientswith chronic lymphocytic leukaemia, patients withthis condition were included in the investigation.

Special emphasis has been given to the folicacid absorption test, as Chanarin, Mollin, andAnderson (1958) reported that injected folic acidwas cleared from the plasma more rapidly thannormal in some patients with leukaemia andrelated disorders. These patients had an associatedmegaloblastic anaemia, and it was considered thatthe anaemia was due to folic acid deficiency.Proliferating neoplastic cells have greater thannormal requirements for folic acid (Swendseid,Bethell, and Bird, 1951); however, Chanarin andhis colleagues noted that some patients failed toabsorb folic acid normally.The present paper describes in detail the folic

acid absorption test used in this laboratory, andreports the results of several intestinal absorptiontests in patients with lymphosarcoma and relatedconditions, comparing these to folic acid absorp-tion and the clinical state of the patient.

MethodsFolic Acid Absorption Test.-The folic acid absorp-

tion test was performed as follows.

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Preparation of the Subject.-The subjects wereloaded with folic acid before the test. This wasachieved in normal control individuals by giving15 mg. folic acid orally on each of the fourth, third.and second days before the test. In patients in whomthe absorption of folic acid was to be studied, 15 mg.sodium folate (" folvite," Lederle) was given by sub-cutaneous injection on each of these days. No folicacid therapy was given the day before the test.Subjects fasted from nine hours before until after the

completion of the test. During this time water wasallowed; food and liquids other than water may affectserum turbiditv readings and were withheld.

Test Procedure.-Five millilitres of venous bloodwere withdrawn from the fasting subject and allowedto clot in a sterile tube at 37° C. A solution ofsodium folate in 20 ml. of water containing the equi-valent of 2 mg. folic acid was then drunk. This solu-tion was prepared from an ampoule of sodium folatecontaining the equivalent of 15 mg. folic acid/ml.

TABLE ICYTOLOGICAL, HAEMATOLOGICAL, AND CLINICAL FINDINGS

TotalHaemoglobin Leucocytes(g./100 ml.) (thousands!

c.mm.)

6 5

15-2

11-3

9-1

9-1

12 5

12-6

10-1

13-1

15-2

9-1

11-1

111

10-3

109

11 5

10*1

12-2

8-9

11-3

9-1

4-4

5-3

190

2-2

6-0

8-4

1-5

3-5

129

92

600

129

211

160

84

137

122

6-4

1.9

12-0

6-6

Differential White BloodCount (%) Clinical Features

P L M Abn. Other

37 58 2

57 31 11

26 63

49 31

20 66

41 46

3

14

10

6

50 24 26

66 24 10

2

96

97

98

3

3 I 97 _-

99

8 91

93

79

83

12

94

53

91

97

8

8

59141

4

37

10

8

12

2

8

7

4

7

Occas.

Occas.

17

3

3

2

Weight loss. Haemolytic anaemiacorrected by splenectomy. Occa-sional diarrhoea

Weight loss. Generalized lymphadeno-pathy including abdominal masses.Anorexia, vomiting, abdominal pain

Lymphadenopathy and hepatospleno-megaly. No gastrointestinal symp-toms

Weight loss. Lymphadenopathy andhepatosplenomegaly. Abdominalmasses. Occasional diarrhoea. As-cites and pleural effusion

Weight loss. Splenomegaly. Recurrentmelaenal diarrhoea. Staphylococcalskin infections

Weight loss. Lymphadenopathy andsplenomegaly. No gastrointestinalsymptoms. Recurrent infections

Weight loss. Abdominal and media-stinal lymphadenopathy. No gastro-intestinal symptoms. Pleural effusion

Weight loss. Hepatosplenomegaly.Abdominal distension with colickypain and recurrent melaenal diar-rhoea. Osteoporosis and vertebralcollapse

Weight loss. Massive lymphadenopathyinguinal region with oedema of leg.No gastrointestinal symptoms

Weight loss. Generalized lymphadeno-pathy. Splenomegaly. Abdominalmasses. No gastrointestinal symptoms

Weight loss. Generalized lymphadeno-pathy. Hepatosplenomegaly. Ter-minal melaenal diarrhoea. Weakness

Weight loss. Generalized lymphadeno-pathy. Hepatosplenomegaly. Ab-dominal distension and flatulence

Weight loss. Generalized lymphadeno-pathy. Hepatosplenomegaly. Occa-sional diarrhoea. Recurrent infections

Generalized lymphadenopathy. Hepato-splenomegaly. No gastrointestinalsymptoms. Recurrent infections

Weight loss. Splenomegaly. Recurrentdiarrhoea. Recurrent infections

Splenomegaly. No gastrointestinalsymptoms

Weight loss. Abdominal masses.Nausea and vomiting following radio-therapy. Recurrent infections

Weight loss. Abdominal masses. Nogastrointestinal symptoms, exceptdiarrhoea following radiotherapy

Splenomegaly. Infiltration stomach,ileum, large bowel at necropsy.Diarrhoea. Anaemia

Weight loss. Diarrhoea. Abdominaldistension. Pigmentation

Weight loss. Abdominal pain of ulcertype. Achlorhydria. Recurrent infec-tions. Anaemia. Previous nephrec-tomy

Patient Age CytologicalDiagnosisSex

M

M

M

F

As

Bo

Co

Ev

Fr

Mp

Mk

Mo

Ba

Be

BI

Go

Md

Ca

Ho

Cr

Ct

Kr

We

Do

Wo

58

54

68

63

72

50

42

62

84

66

38

72

65

68

75

79

21

50

34

54

74

Chronic lympho-cyticleukaemia.. ..

Hodgkin'sdisease

,,

Reticulumcell sarcoma

Whipple'sdisease

Macroglobulin-aemia

- I_Ij II

441

I

I

I

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TABLE 1IOTHER LABORATORY AND RADIOLOGICAL FINDINGS

Pat

Norval

AB

C

iet SeutSrmalla Daily Percentage Folictient Serum Serum rum Pla,,a Faecal Fat Fat in Xylose AciPoen

Albumin Globulins Caroteine ExcrtiorFacesAciProtein Excretion Faeces ~Absoptio Absorption

Greater Serumrmal 6 0-7 5 3 5-5 5 1-5-3-5 Over 70 Less than Less than than 4 g. in Peaklues g. l100 ml. g./ lOO ml. g. I100 ml. mg. lO00 ml. 5 g. IDay 25% 5-Hour 70-130

Urine pmg. ml.ks 6-2 3-3 2 9 - 6-7 - - 483o 6-4 2-9 3.5 41 - 6-9 65

ro 6 6 3-1 3.5 157 lo 60EvFr

MpMkMo

BaBe

BIGo

Md

Ca

HoCrCtKrWe*Do

Wo

4.56-8

6-06 47.9

5 6

5.969

5.5

5.7

6-36-98-06-9

5 0

6-9

2 22-3

222-7

8

2-2I_

3 64-4

2-6

3-2

3-02-9293-4

2-3

2-6

2-3 534.5 60

3-8 16

3.7 276 1 27

3-4 83

2-3 -2 5

2 9

2 5

3-34-05-13.5

2-7

4.3

56

42

52

241232233

3

155

1-9

6-2

16

6227

32

6-4

36

49

9

7-21525 228

2 5 105*9 28

5.76-32-6

Inc

188238

25 20- 12

- 242-9 66

2 1 20

5.7 182

ontinent 322-3 1165.3 444-8 35

82-1 52

3-8 180

58Co-B,2Absorp-

tion

UrinaryExcre-tion

16-36%

19 219-326-3

RadiologicalFindings

Stomach normall Mucosal thickening of

stomach. Normaljejunal pattern

Duodenal diverti-culum. Abnormaljejunal pattern

4-7 -

14-4 Normal stomach andjejunal pattern.Gross osteoporosis

Normal stomach andjejunal pattern

Normal stomach andjejunal pattern

6 7 Normal jejunalpattern

- Normal stomach andjejunal pattern

241

4.5Normal stomach

Normalt Abnormal jejunalpattern

28-6 Normal sto mach andjejunal pattern

* This patient had been receiving oral folic acid therapy for a month before the test. tFaecal excretion test used.oral dose 58Co-Bst was absorbed.

0-68 /tg. of a I l'g.

The folic acid content of an ampoule of thesame batch number had been checked previously byassaying against a preparation of crystalline folic acid(Light) stated to be at least 990o pure. Blood sampleswere withdrawn at hourly intervals for three hours.Serum from all four blood samples was stored at-20° C. until tested for folic acid content.Assay of Folic Acid.-Serum folic acid activity was

estimated microbiologically using a mutant strain ofStreptococcus lactis R (A.T.C.C. 8043). The mutantstrain is approximately three times as sensitive tofolic acid as the standard strain. The technique usedwas essentially that of Teply and Elvehjem (1945).Growth turbidity was recorded with an EEL nephelo-meter.

Other Tests Used.-Other tests included radioactivevitamin B12 absorption (Pitney and Stokes, 1958),xylose absorption (Benson, Culver, Ragland, Jones,Drummey, and Bougas, 1957), and estimations offaecal fat, serum proteins and plasma carotene bystandard methods. Fat balances were estimated bythe method of Frazer (1956). In some patients radio-logical examination of the stomach and small bowelwas also performed. In a few patients estimationsof serum calcium and inorganic phosphate wereobtained.

Subjects StudiedFolic acid absorption tests were carried out on 19

normal subjects.A combination of tests, including the folic acid

test in all instances, was performed on eight patientssuffering from lymphosarcoma, eight suffering fromchronic lymphocytic leukaemia, and five with miscel-laneous related conditions. Brief clinical andhaematological notes on these patients are includedin Table I, and the relevant laboratory results inTable ll.The diagnosis of lymphosarcoma was proved in all

cases by histological examination of biopsy material.In five patients the peripheral blood was free ofabnormal cells; three showed some abnormal cells,but the appearances were not those of lymphocyticleukaemia. One patient (As) had significant anaemia,which was due to splenic hypersequestration andresponded to splenectomy. Clinically, seven of theeight had recent weight loss, and five symptoms refer-able to the gastrointestinal tract. In one (Bo) theseconsisted of anorexia and pain suggestive of pepticulcer. However, he had no diarrhoea or intestinalcolic. Two (As and Ev) had mild, transitory boutsof diarrhoea, and in a third (Fr) there was a pasthistory of recurrent melaena. In one patient only

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(Mo) were bowel symptoms prominent; she hadrepeated episodes of diarrhoea with melaena requiringblood transfusion.The eight patients with chronic lymphocytic leuk-

aemia all had blood counts typical of this condition(Table I). Six had recently lost weight, two (Md andHo) had mild transient diarrhoea, and one (Go)abdominal distension with flatulence and vagueabdominal pain. Melaena was present in one patient(Bl) who developed terminal melaenal diarrhoea.The five patients with miscellaneous related condi-

tions comprised two patients suffering from Hodgkin'sdisease, and one each with reticulum cell sarcoma,Whipple's disease, and macroglobulinaemia. Bothpatients with Hodgkin's disease had recent weight loss,but no gastrointestinal symptoms except transientnausea and vomiting in one and diarrhoea in theother following radiotherapy. Diarrhoea was aprominent symptom of the patient with reticulum cellsarcoma. The patient with Whipple's disease is tobe reported in full elsewhere (Maddocks, Ralston, andWeiden, 1960). This patient had clinical malabsorp-tion and steatorrhoea. The diagnosis was establishedby peroral suction biopsy of the small bowel. Thepatient with macroglobulinaemia has been includedin this series as this condition is thought by some torepresent a variant of lymphosarcoma, although shehad no clinical signs of this disease. Details of thispatient have been published previously (Pitney,O'Sullivan, and Owen, 1958).No patient in any group had clinical or radio-

logical evidence of primary intestinal tumour,although some had palpable masses due to enlargedmesenteric lymph nodes. One patient (Bo) had radio-graphic appearances suggestive of gastric lympho-sarcoma, but these appeared late in his disease, andthere were no signs of small bowel infiltration. Noneof the patients had associated megaloblastic anaemia.They were studied at a random time during the courseof their disease, but wherever possible when theywere not receiving active treatment. Some had, how-ever, received prior treatment with nitrogen mustard,"chlorambucil," or radiotherapy.

Finally, folic acid absorption tests were alsocarried out on four patients suffering from extensivemetastatic carcinoma of non-alimentary origin. Thesepatients were not studied fully, and are not discussedin detail below.

Results(1) Folic Acid Absorption Tests.-The results

are as follows:Normal Subjects. - Measurable folic acid

activity could be demonstrated in every serumsample obtained from 19 normal adults for threehours after an oral dose of sodium folate contain-ing the equivalent of 2 mg. folic acid. In 17, thepeak serum concentration was found at the end ofthe first hour. In the remaining two subjects, thepeak occurred at the second hour. Serum concen-trations were falling rapidly by the third hour.

0120E

'*~~10z0

80zu 600

40

Lu

0 1I 2 3TIME AFTER ORAL DOSE (HOURS)

FIG. 1.-Serum folic acid concentrations in 10 normal subjectsfollowing a standard oral dose of sodium folate containing theequivalent of 2 mg. folic acid.

TIME AFTER ORAL DOSE (HOURS)FIG. 2.-Serum folic acid concentrations in three normal subjects

who received one half the standard dose. The shaded arearepresents the normal range of values obtained with the standardoral dose.

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444W. R. PITNEY, R. A. JOSKE, and NORMA L. MACKINNON

The peak concentrations attained ranged from 70to 130lumg. folic acid per millilitre of serum.

140 Fig. 1 shows a selection of 10 normal folic acidabsorption curves which are representative of thegroup. This selection includes the highest and

E 120 lowest curves obtained in the 19 normal subjects.11.% ////Measurable serum activity2

I^ t///// obtained in normal subjects after 1 mg. orally,I10 but the curves were flatter (Fig. 2). The three

////////// subjects shown in this figure were tested subse-

80 80 quently with the 2 mg. dose and the absorption!R }////j7v//////^curves fell within the limits shown in Fig. 1. After

a - //,~/j7vi//// these experiments, a standard dose of sodiumw 60 } folate containing the equivalent of 2 folic

Zu VA&// ///^///was used in all instances. A peak serum concen-

<>404 tt W_ 4 tration less than 60y umg./ml. was consideredU 40 abnormal.

32 fJ%/// ZPatients with Lymphosarcomna.-In are

cc20

shown the absorption curves given by the eight

patients suffering from lymphosarcoma. Five ofthe eight patients showed subnormal peak serumfolic acid concentrations. Four of the five

0 1 2 3 patients with possible small bowel involvement on

TIME AFTER ORAL DOSE (HOURS) clinical grounds showed subnormal peak concen-FIG. 3.-Serum folic acid concentrations in eight patients with trations, as did five of the seven with recent

lymphosarcoma. significant weight loss.Patients with Chronic Lymphocytic Leukaemia.

-The absorption curves of the eight patients withchronic lymphocytic leukaemia are shown in Fig.

*,182,U mg 4. Five showed subnormal peak serum concentra-140 tions. These five all had significant weight loss.

Three of the four patients with symptoms sugges-E \ tive of small bowel involvement produced abnormal

12 curves. One patient (Ca) had an abnormally highE peak serum concentration. She was receiving

%=-.7 t/treatment for a urinary tract infection at the time10 of investigation. This is discussed more fully

Z below.

$80 t///////x/X\ Patients with Miscellaneous Conditions.-The6-cc VV////////Xabsorption curves of these patients are shown in

60 Fig. 5. The two patients with Hodgkin's disease

z 60 both showed subnormal peak serum concentra-OK tions. One of these patients (Kr) was, however,

U receiving radiotherapy to the abdomen at the time40 of the test for para-aortic lymphadenopathy and

had temporary diarrhoea. Both were losingw

20 /,// _ _ _ = weight steadily. The absorption curve of patient

(//20 We was very flat with a peak serum concentra-

tion of 8 timg./ml. This patient had moderatediarrhoea and postrmortem examination later

0 1 2 3 showed extensive infiltration of the bowel withTIME AFTER ORAL DOSE (HOUR$) neoplastic cells. The patient with Whipple's

FiG. 4.-Serum folic acid concentrations in eight patients with disease (Do) also showed a subnormal peak

chroniclymphocyticleukaemia. serum concentration. He had steatorrhoea and

true clinical malabsorption syndrome. The

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/-

E

Il-

z

vz

n

0

U

0 12 3

TIME AFTER ORAL DOSE (HOURS)FIG. 5.-Serum folic acid concentrations in patients with Hodgkin's

disease (0-0), reticulum cell sarcoma (0-0), Whipple'sdisease (-A), and macroglobulinaemia (A-A).

patient with macroglobulinaemia (Wo) had an

abnormally high peak serum concentration. It isof interest that she had undergone nephrectomysome years previously.The four patients with disseminated carcinoma

all showed peak serum folic acid concentrationswithin the normal range.

(2) Other Investigations.-Results of the otherinvestigations suggested frequent gastrointestinalinvolvement in the patients studied.The serum albumin concentration was below

3.5 g. per 100 ml. in 17 of 19 patients studied,and below 3.0 g. per 100 ml. in 12 of these. Allpatients with lymphosarcoma had some hypo-albuminaemia, as did five of the seven withchronic lymphocytic leukaemia. No patient inany group revealed hypoglobulinaemia, butincreased serum globulin was present in four ofeight with lymphosarcoma, and one patient eachwith chronic lymphocytic leukaemia, Hodgkin'sdisease, and macroglobulinaemia. This frequentoccurrence of hyperglobulinaemia meant that,despite the frequent low serum albumin concen-

trations, the total serum protein levels were below6.0 g. per 100 ml. in only six of 19 patients, fourof whom were suffering from chronic lymphocyticleukaemia. Statistical analysis showed no signi-ficant relation between serum albumin or globulin

levels and low peak serum folic acid concentra-tions, but the association between hypoprotein-aemia and diminished folic acid absorption wassignificant at the 5% level.

Diminished fat absorption was suggested in 13of 17 patients by low plasma carotene concen-trations (less than 70 juLg. per 100 ml.), andbiochemical steatorrhoea was shown in seven of12 by increased faecal fat contents in randomfaecal specimens. Complete six-day fat balancesconfirmed steatorrhoea in three of four patients.The patient with a normal fat balance (Mo) was,however, unable to tolerate a diet containing 50 g.fat daily, and this result must therefore be con-sidered equivocal. The three groups of patientsdid not differ significantly in these results, nor wasthere significant correlation between either lowplasma carotene levels or high faecal fat contentsand low peak serum folic acid concentrations.

Xylose absorption tests were performed on 15patients, and impaired carbohydrate absorptiondemonstrated in eight. The results showed somedifferences in the three groups of patients. Onlytwo of six with lymphosarcoma had abnormalxylose absorption tests, while abnormal xylosetests were shown in four of five with chroniclymphocytic leukaemia. There was again nosignificant correlation between xylose absorptionand peak serum folic acid concentrations.The other investigations may be mentioned

briefly. Four of 10 patients investigated werefound to have defective 58Co-B12 absorption. Theradioactive vitamin B12 was given orally withoutintrinsic factor and the defective absorption couldhave been due to either deficiency of endogenousintrinsic factor secretion or to defective absorptionof the vitamin B12-intrinsic factor complex. Onlyone patient of seven studied (Do, with Whipple'sdisease) had hypocalcaemia. Radiological studiessuggested lymphosarcomatous infiltration of thestomach in one patient (Bo), although he had anormal small bowel pattern. The typical segmen-tation pattern of the malabsorption syndrome wasseen in only two of nine patients, one with lympho-sarcoma and one with Whipple's disease.Comparison of the folic acid absorption test

with the other biochemical parameters used isdifficult, for in these patients, as is usually the casein studies of intestinal absorption, the biochemicalprofiles varied in apparently random fashion frompatient to patient. In general terms, however, thefolic acid absorption test appeared to be the mostsensitive of the methods used in this study, and inall patients with low peak serum folic acid con-centrations collateral evidence of malabsorptionwas obtained, and such patients tended to show

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evidence of multiple absorptive defects. Thus,all patients, except Ca, with a total serum proteinconcentration below 6.0 g. per 100 ml. hadabnormal folic acid absorption tests. Of the 21patients studied in detail, low peak serum folicacid concentrations were observed in 14, whilehypoproteinaemia was found in six of 19, bio-chemical steatorrhoea in nine of 14, and depressedxylose absorption in eight of 15. Low plasmacarotene levels were frequent, but difficult tointerpret in patients who were frequently anorexic.

DiscussionThe folic acid absorption test described in this

paper differs in some respects from that reportedby Chanarin, Anderson, and Mollin (1958). Weconsider it important to fast the patient in the strictsense. Even the amount of milk contained in acup of tea may alter the turbidity of serum asrecorded by a nephelometer, and the usual lightbreakfast containing buttered toast may causenaked-eye turbidity of serum. The inclusion ofa preliminary serum blank in the assay of testserum samples allows correction to be made fornon-specific turbidity. Chanarin and co-workersused a test dose of crystalline folic acid dissolvedin dilute alkali. Folic acid is likely to be pre-cipitated out of this solution in the acid milieu ofthe gastric contents. "Folvite " appears to bemore stable at low pH and is probably absorbedmore rapidly. This may explain why 17 of the 19normal subjects reported in this paper showedpeak serum levels after the first hour, whereas thecontrols of the workers mentioned above showedpeaks mostly at the second hour. A 2 mg. doseof folic acid as " folvite " gave similar serumvalues to those described by Chanarin et al. usinga 3 mg. dose of folic acid dissolved in dilutealkali. A 2 mg. oral dose gave more satisfactorycurves than a 1 mg. dose (Fig. 2).The serum concentration of folic acid in these

tests is influenced by the amount of folic acidabsorbed from the intestine during the time oftesting, the rate of deviation to the tissues and therate of renal excretion of folic acid. In renalfailure delayed excretion of folic acid might beexpected and we have found abnormally high peakserum concentrations in patients with uraemia.Patient Ca (Fig. 4) was suffering from pyelo-nephritis at the time the test was performed, andpatient Wo (Fig. 5) had undergone a previousnephrectomy. Coexisting renal disease is prob-ably a contraindication to this test.The possibility has been considered that the

subnormal folic acid absorption curves observed

in this investigation were due to increased tissuerequirements for folic acid rather than defectiveabsorption. Pollack, Taylor, and Williams (1942)found higher amounts of folic acid in varioustypes of neoplastic tissue than in the correspond-ing normal tissue, and Swendseid et al. (1951)found higher concentrations of folic acid inleukaemic leucocytes than in normal white cells.Increased tissue requirements does not seem asatisfactory explanation since the four patientswith disseminated carcinoma tested presumablyalso had increased tissue demands for folic acidbut all showed normal absorption curves.

Inadequate loading with folic acid before theabsorption tests is an unlikely explanation for thesubnormal curves. Urinary excretion of folicacid was measured in two normal subjects whoeach received injections of 15 mg. folic acid onthree successive days. This is the loading pro-cedure used in preparation of patients for the tests.Total three-day urinary excretion values in thesenormal subjects were 17.9 and 19.3 mg. respec-tively. Assuming that biliary or intestinal loss offolic acid was not significant, these subjectsretained more than one half of the total 45 mg.folic acid injected. The highest concentration oftotal apparent folic acid activity in the body isstated to be in the liver and the concentration inthis organ is normally in the range from 1.3 to5.8 ug./g. wet tissue (Girdwood, 1952, 1953). Anormal liver thus contains no more than 10 mg.material with folic acid activity. The loadinginjections would be expected to more than doublethe normal body stores.

Finally, the results of other absorption tests inthese patients support the view that the low folicacid curves were indeed the result of impairedintestinal absorption, and suggest further that thefolic acid absorption test may be the most sensitivesingle test of intestinal absorption, at least in thisgroup of patients. It would therefore appzar thatunselected patients suffering from lymphosarcomaand chronic lymphocytic leukaemia commonlyshow laboratory evidence of intestinal malabsorp-tion. In the majority of our patients there wereno clinical signs of severe gastrointestinal involve-ment. Certainly the typical sprue syndrome wasseen in only one patient. Malabsorption couldexplain some of the puzzling clinical featuresassociated with terminal lymphosarcoma. Ascitesand oedema of the lower extremities are oftenascribed to pressure from enlarged abdominallymph nodes. In these patients hypoproteinaemiais common and impaired protein absorption orexudative enteropathy (Gordon, 1959) might offeran alternative mechanism. We have found that

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such patients are often improved after intra-venous therapy with salt-poor concentrated serumalbumin. Weight loss, which is often a prominentfeature in both lymphosarcoma and chroniclymphocytic leukaemia, is also common withintestinal malabsorption. The occasional develop-ment of megaloblastic anaemia in these patientscould be due to malabsorption of folic acid aswell as to the increased demands of proliferatingneoplastic tissue for folic acid.

We wish to thank Sister A. Smith and Dr. M. F.Carruthers for their help in the preparation of thepatients, and Mr. R. E. Davis and Mr. P. Onesti fortechnical assistance with the microbiological assays.Dr. D. H. Curnow carried out the biochemicalestimations.

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G. D., and Bougas, E. (1957). New Engl. J. Med., 256, 335.Chanarin, I., Anderson, B. B., and Mollin, D. L. (1958). Brit. J.

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