Journal of Clinical InvestigationVol. 41, No. 5, 1962

ANTIBODY TO INTRINSIC FACTORIN SERUMFROMPATIENTSWITH PERNICIOUS ANEMIA*

By GRAHAMH. JEFFRIES,t DONALDW. HOSKINS AND MARVINH. SLEISENGER

(From the Department of Medicine, New York Hospital-Cornell Medical Center,New York, N. Y.)

(Submitted for publication October 30, 1961; accepted February 1, 1962)

Schwartz (1, 2) and Taylor (3) have suggestedthat sera from patients with pernicious anemia maycontain antibody to intrinsic factor. Both inves-tigators studied the interaction of serum and in-trinsic factor in these patients by measuring vita-min B12 absorption after oral test doses of cobalt°0-labeled vitamin B12 (Co60B,2) mixed with hogintrinsic factor and serum (1-3). In these tests,sera from 40 to 50 per cent of the patients withpernicious anemia inhibited vitamin B12 absorp-tion. This inhibition was observed not only withserum from patients who had received oral therapywith intrinsic factor preparations (1, 2) but alsowith serum from those who were untreated andthose who had received only parenteral vitaminB12 (2, 3). Sera from rabbits which had beenimmunized with hog intrinsic factor preparationssimilarly inhibited vitamin B12 absorption (4).

Immunological studies by in vitro tanned redcell agglutination techniques have yielded positiveagglutination reactions between pernicious anemiasera and tanned red cells coated with hog intrinsicfactor (2, 5). However, in correlating this invitro test with in vivo absorption studies, Schwartz(2) reported a 33 per cent incidence of false posi-tive or negative agglutination reactions.

While these observations are consistent withthe conclusion that specific antibody to intrinsicfactor may be present in the serum of perniciousanemia patients, direct evidence for this is lacking.Although immunological studies in vitro haveshown that sera from pernicious anemia patientsmay react with hog intrinsic factor, a more specificinteraction of gammaglobulin derived from perni-cious anemia serum and purified human intrinsicfactor has not been demonstrated.

The experiments described here were carriedout in an attempt to define more precisely the in-

* Supported in part by United States Public HealthService Grant C-9386.

t USPHSResearch Career Development Awardee.

teraction of pernicious anemia sera and human in-trinsic factor both in vivo and in vitro. The block-ing action of pernicious anemia sera on intrinsicfactor activity was demonstrated by vitamin B12absorption tests in which mixtures of serum withintrinsic factor-Co60B12 complex were fed to pa-tients with pernicious anemia (2). The in vitrointeraction of sera and intrinsic factor was studiedby electrophoretic and immunologic techniques.It was found that the pernicious anemia sera thatinhibited intrinsic factor activity in absorptionstudies contained y-globulin which combined withhuman intrinsic factor-Co60B,2 complex.

METHODS

PatientsSerum was obtained from 20 normal medical students,

44 patients with pernicious anemia, and 5 patients withatrophic gastritis without pernicious anemia. The diag-nosis of pernicious anemia had been established by dem-onstration of: a) a macrocytic anemia with megaloblasticbone marrow; b) gastric achlorhydria on maximal his-tamine stimulation (6); and c) subnormal absorption ofCoOB,2 which was corrected by intrinsic factor. Serafrom pernicious anemia patients were obtained beforetreatment (6 patients) or during maintenance therapywith parenteral injections of vitamin B12 (38 patients).Those patients with atrophic gastritis without perniciousanemia exhibited hypochlorhydria on maximal histaminestimulation but absorbed Co'B,2 normally.

Twenty patients with pernicious anemia and 1 patientwith atrophic gastritis without pernicious anemia (Case7) were selected for Co°B12 absorption studies with in-trinsic factor and serum. This selection was in part in-fluenced by the results of in vitro studies. This study in-cluded 10 patients whose serum altered the electropho-retic mobility of an intrinsic factor-Co°B,2 complex.

Preparation of human intrinsic factor-Co80B,2complexCobaltr-labeled vitamin B12 (Co"B,2) , of specific ac-

tivity 1,000 ,uc per mg, was added to gastric juice freshlycollected from normal medical students during histaminestimulation. The concentration of added Co'B,2 (250 m/ug

1 Obtained from Abbott Laboratories, Oak Ridge, Tenn.

1106

ANTIBODY TO INTRINSIC FACTORIN PERNICIOUS ANEMIA

per ml of gastric juice) exceeded the binding capacity ofthe gastric juice so that vitamin B.,-binding componentswere saturated (7). This mixture was dialyzed againstwater, lyophilized, redissolved in a small volume of 0.03M borate buffer at pH 9.0, and fractionated by electro-phoresis on starch gel (8).

The anodally migrating zone of bound Co'B,,, wasidentified and quantitated by counting 0.5-cm segmentsof the electrophoretic strip in plastic tubes in a well-typescintillation counter against a Co' standard. Those seg-ments of the electrophoretic strip containing bound Co'B12were homogenized in a small volume of distilled waterto give a final concentration of approximately 100 mtg ofvitamin B,2 (100 muc Co'B,2) per ml of homogenate, andstored at - 200 C.

Studies using this biologically active bound Co'B12complex (IF-CoeB,,) (8), offered three advantages overprevious investigations with hog intrinsic factor prepara-tions (1-3, 5). 1) Homologous (human) intrinsic fac-tor was utilized; autoantibody, reacting with human in-trinsic factor, might not be detected in studies with heter-ologous intrinsic factor. 2) The sensitivity of tests todemonstrate the interaction of intrinsic factor and serumcomponents is maximal when labeled intrinsic factoralone, IF-Co'B,,, is present in the system. In previousstudies with hog mucosal preparations, an excess ofunlabeled intrinsic factor would have reduced the sensi-tivity of the tests (1-3). 3) IF-Co'B,2 complex preparedby starch gel electrophoresis of human gastric juice (8)is contaminated only by those gastric juice componentsthat have similar electrophoretic mobility. In contrast tothis, hog intrinsic factor preparations are electropho-retically heterogeneous (9). Both hog intrinsic factorpreparations, and the IF-Co°'B, complex used in thisstudy, however, may contain vitamin B,2-binding sub-stances other than intrinsic factor.

Absorption studiesVitamin B,, absorption tests were carried out on 21

patients (Figure 1) by a modification of the method ofSchilling (10). Urine was collected for 48 hours afteroral test doses containing 200 m/tg of Co'B,, (200 mlAc).Flushing doses of unlabeled vitamin B:, (1,000 lsg) wereinjected 1 and 24 hours after the test dose. The 48-hoururinary excretion of Co'Bl,, expressed as a percentage ofthe administered dose, was greater than 10 per cent in thenormal subject.

Successive tests were a) Co'Bl2 alone; b) IF-Co'B,(containing 200 m/Ag vitamin B12); c) IF-Co'"B,, + 10 mlof normal serum; and d) IF-Co`B% + 10 ml of the pa-tient's own serum. The tests were carried out at weeklyintervals in each subject. In half of the patients stud-ied, test d preceded test c. In a few patients the absorp-tion studies were confined to tests a and d.

In vitro studies on the interaction of sera andserum protein fractions with IF-Co6B 12

was mixed with 0.1 or 0.2 ml serum from 20 normal sub-jects, from 44 patients with pernicious anemia, and from5 patients with atrophic gastritis without perniciousanemia. This mixture was introduced into a transverseslot in a starch gel electrophoretic strip, and electropho-resis was carried out at 100 C for 16 hours at constantvoltage of 5 v per cm (11). In simultaneous control ex-periments, mixtures of 10 m~ug Co'B,, with 0.2 ml serumwere separated. The distribution of radioactivity wasmeasured by counting 0.5-cm segments of the gel strip ina well-type scintillation counter.

In a single experiment, varying volumes of serum(0.025 to 0.2 ml) from Patient 1 were mixed withIF-Co'B,2, containing 10 mfsg vitamin B12, and wereseparated by starch gel electrophoresis.

Gammaglobulin was prepared from normal and per-nicious anemia sera by starch gel electrophoresis (11).At pH 8.6, the y2-globulin fraction migrated cathodally.Segments of starch gel electrophoretic strips containingthis protein fraction were mixed with IF-Co'B,2 (10 m/Agvitamin B1,) by grinding the gel with IF-Co'B1, contain-ing homogenate, and the mixture was again separatedelectrophoretically.

B. Paper electrophoresis. This method has been usedpreviously to study the interaction of antisera and labeledantigens (5, 12, 13). In this study mixtures of 0.2 mlserum with IF-CoB,2 (10 mftg of vitamin B,2) wereseparated by paper electrophoresis. Fifty ,ul of this mix-ture was applied to each paper strip (Whatman no. 3)in a Spinco model R paper electrophoretic system, andelectrophoresis was carried out with veronal buffer (pH8.6, 0.075 M) for 16 hours at 250 C. The paper stripswere oven-dried and bisected longitudinally. One halfwas stained with amido black; the other half was cutinto 1-cm segments. The distribution of radioactivity onthe electrophoretic strip was measured by placing thesesegments in plastic tubes in a well-type scintillationcounter.

C. Agar diffusion. These studies were carried out at250 C by the method of Ouchterlony (14), with 1 percent agar in 0.9 per cent saline. Sera from normal sub-jects and from pernicious anemia patients were testedagainst IF-Co'B,1,. In further experiments the agar dif-fusion plates were modified by incorporating Whatmanno. 1 filter paper in the agar gel beneath the diffusionwells. This filter paper, impregnated with agar gel, wasseparated when diffusion was complete, dried at roomtemperature, and placed in contact with radiographicfilm in a cassette. The resulting radioautograph indicatedthe diffusion of IF-Co'B,, in the gel.

RESULTS

In vivo absorption studies of the interaction ofnormal and pernicious anemia sera withIF-Co60B12

A. Starch gel electrophoresis. IF-Co'B,_, (0.1 to 0.2 The results of the absorption studies carried outml of homogenate containing 10 to 30 mlg of vitamin B.) on 21 patients are shown in Figure 1. In 9 pa-

1 107

G. H. JEFEIlRIES, D. WV. HOSKINS AND M. H. SLEISENGER

10

z

0

0o"I

z

E 10

0

zu

t,

S0

T) o

z

7,1

OD

10

A) CO602

B) IF-C060R 12

C) IF-CO60B12 + NORMALSERUM

IFClln PIu rD) IF-C065312 PATIENT'S SERUM.

S.O _

2 3 4 5 6 7

PATIENT MUMBEP8 9 10 11 12 13 14 15 16 17 18 19 20 21

FIG. 1. URINARY EXCRETION OF CO&B12 IN SCHILLINGTESTS ON PERNICIOUS ANEMIA PATIENTS. A) Co'B,2(200 mgg vitamin B,0) without intrinsic factor. B) IF-CoRB,2 (containing 200 m/Ag vitamin B12). C) IF-Co'B,2with 10 ml of normal serum. D) IF-CoGOB,. with 10 mlof the patient's own serum.

The 48-hour urinary excretion of CoB,2 is expressedas a percentage of the orally administered dose. Patient7 suffered from atrophic gastritis without perniciousanemia. Tests on Patients 8 and 9 were carried out with25 and 30 ml of serum, respectively.

tients with pernicious anemia (Cases 1 to 6 and8 to 10) and in Patient 7 with atrophic gastritis,the oral administration of 10 ml of the patients'own serum with IF-CoROB19 inhibited Co60B12absorption. No inhibition was observed whentests wvith normal serum were carried out in thesame patients. In studies on 11 other patientswith pernicious anemia (Cases 11 to 21) the oraladministration of 10 ml of normal serum or of thepatients' own serum with IF-Co60B12 did not in-hibit Co01B12 absorption.

In vitro studies of the interaction of normal andpernicious anemia sera and serum protein frac-tiouis with IF-CoG0B12A. Starch gel electrophoresis of serum with

IF-Co0"B12. IF-Co00Bl.2 mixed with normal se-rum, separated anodally on starch gel electrophore-sis (Figure 2, A). Electrophoresis of IF-Co60B12with pernicious anemia sera that caused inhibitionof C06OB12 absorption (inhibitory sera; Cases 1 to6 and 8 to 10) resulted in the delineation of twozones of radioactivity (Figure 2, B). An anodalzone corresponded to IF-Co0°B12, while the re-maining radioactivity was retained in the zone ofapplication. Serum from Patient 7, with atrophicgastritis without pernicious anemia, gave a similardistribution of radioactivity. Sera from 35 pa-

ELECTROPHORETICMIGRATION OF INTRINSIC FACTOR - C00B12 COMPLEXWITH

PERNICIOUS ANEMIA SERA

30

a20

u

0

10

0

Z '..,

a-~z

.0'oC0o

A ) NORMALSERUM

-i J-.-

3 2 v-7

C'.'I:-'C-Tlt' st ''I"I-T

FIG. 2. STARCH GEL ELECTROPHORESISOF NORMALANDPERNICIOUS ANEMIA SERA WITH IF-Co°B,2. Starch gelelectrophoresis of mixtures of serum (0.2 ml) withIF-Co'B,2 (containing 30 mpig vitamin B12). The distri-bution of radioactivity and a corresponding electropho-retic strip stained with amide black lOB are shown. A)Normal serum. B) Pernicious anemia serum that in-hibited vitamin B,2 absorption (Patient 1 of Figure 1).

1 1()

ll-.-

Ills I a

..

ANTIBODY TO INTRINSIC FACTORIN PERNICIOUS ANEMIA

tients with pernicious anemia, including those thatdid not inhibit Co6°B12 absorption (Cases 11 to21), caused no alteration in the electrophoreticmigration of IF-Co °B1,, no radioactivity being re-

tained in the zone of application.B. Starch gel electrophoresis of serum with

Co060B12 without intrinsic factor. Starch gel elec-trophoresis of mixtures of serum with Co°0B,2separated the radioactivity in a single cathodalpeak with a mobility corresponding to that of free(nonprotein-bound) Co°0B2. The distribution offree Co00B12 overlapped that of y-globulin. Lessthan 2 per cent of the added Co06B12 (i.e., lessthan 1 mjug of vitamin B12 per ml of serum)migrated anodally or was retained in the zone ofapplication. This distribution was similar for bothnormal and pernicious anemia sera (Figure 3).

C. Starch gel electrophoresis of serum proteinfractions with IF-Co80B1,. Gammaglobulin, sep-

ELECTROPHORETICMIGRATION OF CO60B12 WITH PERNICIOUS ANEMIA SERA

80

A) NORMALSERUM

<040.

Z80 | ' ~~~~~B)PERNICIOUS ANEMIA

Z60 ' ' ~~~~~SERUM

60

0

0Q20 rJ

4 3 2 1 V 1 2 3 4 56 7

CATHODE ORIGIN ANODE

MIGRATION DISTANCE (CM)

FIG. 3. STARCHGEL ELECTROPHORESISOF NORMALAND

PERNICIOUS ANEMIA SERA WITH CoeB1, (WITHOUT IN-

TRINSIC FACTOR). Starch gel electrophoresis of mixturesof serum (0.2 ml) with Co'Bl2 (10 mag vitamin B12).

The electrophoretic distribution of radioactivity is shown.A) Normal serum. B) Pernicious anemia serum thatinhibited vitamif B12 absorption (Patient 1, Figure 1).

ELECTROPHORETICMIGRATION OF INTRINSIC FACTOR- C060B12 COMPLEXWITH

SERUM PROTEIN FRACTIONS

60

50

40

30

20

10

60

50

t 40

-° 20

0

40

O 30

Z 20

0 5

0

I-

n

40

u

50

4e 0

20

20

:) 10

B) GAMMAGLOBULIN(PERNICIOUS ANEMIA)

C) GAMMAGLOBULIN(PERNICIOUS ANEMIA)

4 3 2 2 3

CATHODE ORIGIN

MIGRATION DISTANCE (CM)

D) ALBUMIN + ALPHA ANDBETA GLOBULIN(PERNICIOUS ANEMIA)

4 5 6 7 8 9ANODE

FIG. 4. STARCH GEL ELECTROPHORESIS OF IF-Co1*B1.2WITH SERUMPROTEIN FRACTIONS FROMNORMALAND PER-

NICIOUS ANEMIA SERA. Serum protein fractions were

prepared by starch gel electrophoresis. IF-CoO°B, (20mtug vitamin B,,) was mixed with the serum proteinfraction and the mixture was again separated by starch

gel electrophoresis. The distribution of radioactivity is

plotted. A) -y-Globulin from 0.2 ml of normal serum.

B) y-Globulin from 0.2 ml of pernicious anemia serum

that did not inhibit Co°'B,2 absorption (Patient 15). C),y-Globulin from 0.2 ml of pernicious anemia serum thatinhibited Co°'B1, absorption (Patient 1). D) a-, p-Glob-

ulin and albumin from the same pernicious anemia serum

as C).

1109

I

Y'.. .I

A.,

L-1

G. H. JEFFRIES, D. W. HOSKINS AND M. H. SLEISENGER

A) NORMAAL St RU

-7

B) PERNICIOUS ANEMIASERUM

Ji-h1-

.-.-.-.-.-.--4 3 2 t 2 3 4 i 6 7 8 9

CATHODE OR GIN ANIODE

t. IGRATIO N DISTANCE (CM)

FIG. 5. PAPER ELECTROPHORESISOF NORMALAND PER-

NICIOUS ANEMIA SERA WITH IF-CO'B12. Paper electro-phoresis of mixtures of serum (0.2 ml) with IF-Co'B12(containing 20 mug vitamin B12); 50 of mixture ap-

plied to each electrophoretic strip. The distribution ofradioactivity and a corresponding electrophoretic stripstained with amide black 1OB are shown. A) Normalserum. B) Pernicious anemia serum (with inhibitor;Patient 1).

arated by starch gel electrophoresis from thosepernicious anemia sera that combined withIF-Co60B12 and inhibited Co60B12 absorption(Cases 1 to 10), altered the electrophoretic migra-tion of IF-Co60B12 (Figure 4, C). Radioactivitywas retained in the zone of application. Theanodally migrating protein fractions of the same

sera (albumin and a- and f8-globulins) did notinteract with IF-Co60B12 to alter its electrophoreticmobility (Figure 4, D). Gammaglobulin sep-

arated from normal sera and other perniciousanemia sera was similarly unreactive (Figure4, A and B).

D. Paper electrophoresis of serum withIF-Co010B2. IF-Co60B52 separated anodally withthe electrophoretic mobility of 8-globulin in the

presence of normal serum and noninhibitory per-nicious anemia serum (Figure 5, A). With in-hibitory pernicious anemia serum (Cases 1 to10), IF-Co6°B12 migrated cathodally with they-globulin fraction (Figure 5, B).

E. The interaction of normal and perniciousanemia- sera with IF-Co6B12 on agar diffusionplates. Diffusion of normal or pernicious anemiasera on agar diffusion plates against IF-Co00B12did not result in the appearance of visible precipitinzones in the agar gel. Radioautographs of filterpapers that had been incorporated into the agar dif-fusion plates beneath the well, however, revealedthe presence of linear zones of action betweenpernicious anemia sera and IF-Co0OB12. The zoneof interaction was related only to those sera thatcombined with IF-Co60B12 in electrophoretic stud-ies (Figure 6).

F. The effect of varying serum concentrationson the interaction of inhibitory pernicious anemiaserum with IF-Co60B12. Figure 7 shows the per-centage of radioactivity that was retained in thezone of application when mixtures of IF-Co60B,2(containing 10 mpg of vitamin B12) with varyingvolumes (0.025 to 0.2 ml) of pernicious anemia

A B

0

I cmVI. .

FIG. 6. RADIOAUTOGRAPHOF FILTER PAPER FROM AN

AGARDIFFUSION PLATE. The points A, B, and C indicatethe center of diffusion wells 0.6 cm in diameter. Well Acontained serum from pernicious anemia Patient 1.Well B contained serum from pernicious anemia Pa-tient 2. Well C contained IF-Co'B1, (20 mtg vitaminB12). Diffusion for 3 days at 250 C; radiographic filmexposed for 10 days. Arrows indicate zones of inter-action. Zone of radioactivity encircling well C was an

artifact due to the growth of microorganisms.

90

80

70

60

50

40

30

2 20R.-

Us< o

0

z 080-i070

60

20

z

o 10

IU

1110

ANTIBODY TO INTRINSIC FACTORIN PERNICIOUS ANEMIA

100

SO

40

20

.tI

Ur.

<Ubo1:0,

--u 0.025 0.050 0.075 0.100 0.125

VOLUMEOF PERNICIOUS ANEMIA SERUM (ML)

0.150 0.175 0.200

FIG. 7. STARCH GEL ELECTROPHORESIS OF IF-CO8B,2WITH VARYING VOLUMESOF SERUM. IF-Co'B00 (contain-ing 10 m/Ag vitamin B,2) was mixed with 0.025 to 0.20ml vol of inhibitory pernicious anemia serum (Patient 1,Figure 1), and the mixtures were separated by starch gelelectrophoresis. The percentage of the total radioactivitythat remained in the zone of application is plotted foreach serum volume.

serum from Patient 1 were separated by starchgel electrophoresis. As the volume of serum inthe mixture was increased from 0.025 to 0.125 ml,there was a linear increase in the proportion ofradioactivity retained in the zone of application.With volumes of serum increasing from 0.125 to0.2 ml, there was no further increase in the pro-portion of nonmigrating radioactivity. Thus, inthe presence of an excess of serum there remaineda small proportion of bound Co60B,2 which mi-grated anodally. This nonreacting componentmay represent a bound Co60B12 complex withoutintrinsic factor activity.

The linear increase in nonmigrating radioactiv-ity with increasing amounts of inhibitory serumsuggested a nonreversible binding reaction betweenIF-Co80B12 and y-globulin. This was tested byadding an excess of unlabeled intrinsic factor to amixture of inhibitory serum and IF-Co60B12. Un-labeled intrinsic factor did not displace IF-Co60B12from combination with y-globulin (Figure 8).

G. Quantitation of IF-CoOB152-binding y-glob-ulin in inhibitory pernicious anemia sera. Therelative concentration of IF-Co6OB12-binding y-glob-ulin in inhibitory pernicious anemia sera was cal-culated from the proportion of radioactivity re-tained in the zone of application after starch gelelectrophoresis of mixtures of serum with

IF-Co0°B,2. This is expressed by the equation:X = Y x Z where X represents serum IF-Co80B12binding (mpg vitamin B12 bound as IF-Co60B,2by 1 ml serum), Y represents the percentage ofradioactivity retained in the application zone, andZ represents the concentration of vitamin B12 inthe serum: IF-Co00B12 mixture. Values obtainedfor inhibitory sera, using a single preparation ofIF-Co68B12, are given in Table I (column 1) .

It became apparent during the course of thisstudy that different preparations of IF-Co60B12varied in their content of bound vitamin B12. This

80

70 A

60

50

U 400

.202z

~10

-.

80

70

60B

50

303

20

ZUI.X

3 2 1CATHODE

MIGRATION

4 , 1 :

ORIGINDISTANCE (cm)

2 3 4 5 6ANODE

7

FIG. 8. EFFECT' OF ADDING AN EXCESS OF UNLABELEDINTRINSIC FACTOR (IF) TO A MIXTURE OF SERUMAND

IF-Co'B.2. Serum (0.2 ml from Patient 1) was mixedwith IF-Co'B,2 (containing 5 muog vitamin B50). A)Starch gel electrophoresis of mixture alone; radioactivityremained in or adjacent to the application zone. B)Starch gel electrophoresis of serum: IF-Co'0B.2 mixturewith excess of intrinsic factor. IF, separated from 50 mglyophilized normal gastric juice, was added 10 minutesafter mixing serum with IF-Co'B52. Electrophoresis wasbegun 30 minutes later.

1111

G. H. JEFFRIES, D. W. HOSKINS AND M. H. SLEISENGER

TABLE I

Correlation between inhibition of Co66B12 absorptionand IF-Co&JB12 binding by pernicious anemia sera

1 2 3

Binding oftest dose ofIF-Co6OB12(200 mjugB12) by Inhibitionvol of of CoG°Bi2

Binding of serum absorptionIF-Co6"B12 used in in tests

Patient by serum absorption with pa-no.* in vitro test tient's serum

myg vit. % %B12/mlserum

1 58.0 100 982 28.7 100 983 22.5 100 754 28.8 100 695 31.7 100 776 47.0 100 907 28.8 100 798t 7.0 30 809T 11.3 56 63

10 2.7 14 35

* Patient numbers correspond to those in Figure 1.Schilling tests were performed on this patient using 25 ml of serum.

t Schilling tests were performed on this patient using 30 ml of serum.

variation was due to dissociation of IF-Co60B12complex with loss of Co60B12 during preparativedialysis. The calculated IF-Co60B,2-binding valuesof inhibitory sera thus varied with different prep-arations of IF-Co60B12 (values for serum of Pa-tient 1 from experiments illustrated in Figures 2and 4 and in Table I, respectively, were 28.5, 60,and 58 mug vitamin B19 per ml of serum.

The action between y-globulin of inhibitory seraand IF-Co80B12 in vitro would explain the inhibi-tory effect of these sera in absorption tests if therewas an approximate quantitative correlation be-tween these phenomena. Accurate quantitativecorrelation was not possible because of the vari-able vitamin B12 content of IF-Co60B12 prepara-tions used in both in vitro tests and absorptionstudies.

To quantitate the inhibitory activity of perni-cious anemia sera on vitamin B12 absorption, theurinary excretion of Co80B12 in tests with inhibi-tory sera has been expressed as a percentage ofexcretion in control tests with normal serum (Ta-ble I, column 3). The percentage of the absorp-tive test dose of IF-Co0°B12 (200 mpg vitaminB12) that would have combined with y-globulin inthe inhibitory serum, calculated from the serumIF-Co60B12-binding value (Table I, column 2),was commensurate with the observed inhibitionof vitamin B12 absorption (Table I, column 3).

DISCUSSION

The results of vitamin B12 absorption tests utiliz-ing human IF-Co60B,2 complex mixed with nor-mal and pernicious anemia sera (Figure 1) con-firm the inhibitory effect of some perniciousanemia sera described by Schwartz (1, 2) andTaylor (3). In the present study, inhibition ofvitamin B12 absorption was demonstrated with10 ml of pernicious anemia serum. In Schwartz'sand Taylor's experiments, however, 50 ml of in-hibiting serum was required. This differenceprobably reflects the presence of an excess of in-trinsic factor in the latter studies, rather than adifference in the concentration of serum inhibitor.

Pernicious anemia sera that inhibited vitaminB12 absorption when given orally with IF-Co60B12to patients with pernicious anemia (inhibitorysera) also altered the electrophoretic migration ofIF-Co6°0B2. Onpaper electrophoresis, IF-Co60B12,which migrates anodally in normal serum, mi-grated cathodally with the y-globulin of inhibitorysera. A similar observation was made by Lowen-stein, Cooper, Brunton and Gartha (5) in paperelectrophoretic studies of rabbit immune serummixed with hog intrinsic factor and Co80B2. Onstarch gel electrophoresis, the IF-Co60B,2 complexmigrated anodally in the presence of normal andnoninhibitory pernicious anemia sera, whereas inthe presence of inhibitory pernicious anemia sera,the radioactive complex remained in the zone ofapplication.

Gammaglobulin, separated from inhibitory per-nicious anemia sera by starch gel electrophoresis,was the only serum protein that altered the elec-trophoretic migration of IF-Co60B12. This ex-cludes the possibility that IF-Co0B12 complexmight be combining with unsaturated carrier pro-tein in pernicious anemia serum. It has beenshown that the serum vitamin B12-binding proteinis an a-globulin (15, 16). Furthermore, the ca-pacity of inhibitory sera to bind IF-Co°B12 in they-globulin fraction was considerably in excess ofthe normal vitamin B12-binding capacity of serum(17). Electrophoretic studies with mixtures ofserum and Co6OB12 in the absence of intrinsic fac-tor also exclude the possibility that free vitaminB12 might be combining with a serum componentto form a nonmigrating complex. In these experi-

1112

ANTIBODY TO INTRINSIC FACTORIN PERNICIOUS ANEMIA

ments with free Co60B12, radioactivity was not re-tained in the application zone.

Retention of radioactivity in the zone of appli-cation after starch gel electrophoresis of mixturesof IF-Co60B,2 with y-globulin from inhibitory seraindicates that y-globulin binds IF-Co60B,2 to forma complex which is too large to migrate in thestarch gel medium. The formation of a singleradioactive zone of interaction of inhibitory per-nicious anemia sera and IF-Co60B12 in agar dif-fusion studies, demonstrates that a single complexis formed between IF-Co60B,2 and y-globulin.

On the basis of absorption studies it had beensuggested that antibody present in some perniciousanemia sera might inhibit intrinsic factor activity(2, 3). This inhibitory effect, observed in ourabsorption studies, was confined to sera that re-acted with IF-Co0OB12 upon electrophoresis. Fur-thermore, the amount of IF-Co60B12 that wasbound by inhibitory sera in electrophoresis paral-leled the inhibitory effect of the same sera in ab-sorption tests. This suggests that the inhibitionof vitamin B12 absorption in vivo results from theformation of a complex between IF-Co6OB12 andy-globulin of the inhibitory sera.

The mechanism by which y-globulin of inhibi-tory sera prevents vitamin B12 absorption has notbeen determined. Inhibition occurs, however, onlywhen the blocking y-globulin is present in thelumen of the gastrointestinal tract. Patients withinhibitory sera absorbed vitamin B12 normallywhen IF-Co6"B12 alone was administered orally.Perhaps binding of IF-Co00B,2 to mucosal re-ceptors in the small intestine is prevented by com-bination with y-globulin (2).

The formation of a complex between IF-Co6OB12and y-globulin of inhibitory pernicious anemia serais strong evidence of an antigen-antibody reaction.This is supported by immunological studies in therabbit. Sera of rabbits which had been immunizedwith hog intrinsic factor preparations not only in-hibited intrinsic factor activity in absorption tests(4), but also bound in their y-globulin fraction acomplex of hog intrinsic factor and Co60B12 (5).Proof of the immunological nature of the reactionbetween y-globulin and IF-Co60B12 would be theproduction of a similar human antibody to intrinsicfactor by active immunization, and the observationthat pernicious anemia patients, with inhibitory

sera before immunization, developed an anamnesticresponse.

An autoimmune phenomenon is suggested by thepresence of antibody to intrinsic factor in the se-rum of pernicious anemia patients who have hadno known exposure to exogenous intrinsic factor.The antigenic stimulus may result from the ab-normal release of intrinsic factor from the gastricmucosa into the circulation by mucosal injury, ormay result from an altered responsiveness of anti-body-producing cells to endogenous intrinsic fac-tor. Although this study has more clearly definedthe interaction of pernicious anemia sera and in-trinsic factor, and has indicated the presence ofintrinsic factor antibody in 20 to 30 per cent ofthose sera, the significance of this antibody in thepathogenesis of atrophic gastritis and perniciousanemia is unclear. Chronic atrophic gastritis mayresult from the interaction of autoimmune anti-bodies with antigens in the gastric mucosa. It hasrecently been reported that injections of homol-ogous or heterologous gastric juices into the dogproduce atrophic gastritis (18).

The demonstration of intrinsic factor antibodyin the sera of only 20 to 30 per cent of patientswith pernicious anemia has not been explained.Intrinsic factor antibody, present at one time inthe serum, may disappear when antigenic stimula-tion ceases (with intrinsic factor secretory failure).If this were the case antibody should be demon-strable more frequently in patients who had morerecently developed pernicious anemia, or who suf-fered from atrophic gastritis but had not yet de-veloped vitamin B12 deficiency. In this study onlyone of five patients with atrophic gastritis withoutpernicious anemia, and two of six patients withrecently diagnosed pernicious anemia, showedantibody in their sera. An alternative explanationmay be that atrophic gastritis is the end result ofseveral pathological processes, as yet poorly de-fined, which do not all result in immunologicalphenomena.

Patients with pernicious anemia, who have beentreated by oral administration of hog intrinsic fac-tor and vitamin B12, may become unresponsive tosuch therapy (1, 5, 19). Although Schwartz ini-tially considered that such resistance to oral ther-apy might result from the presence of blockingantibody in the patients' serum, his studies did

1113

G. H. JEFFRIES, D. W. HOSKINS AND M. H. SLEISENGER

not support this hypothesis (1, 2). Lowensteinand co-workers have concluded, however, thatthere may be an immunological basis for acquiredresistance to oral therapy with hog gastric mucosalpreparations in pernicious anemia (5); theseauthors based their conclusion on the results oftanned red cell agglutination tests, correlated withvitamin B12 absorption studies. While hog gastricmucosal preparations were shown to react withsera of resistant patients, human gastric juicewas nonreactive (5). Patients with acquiredresistance to oral hog intrinsic factor absorbedvitamin B12 normally when human gastric juicewas administered. Red cells coated with hogpyloric mucosal extract were agglutinated only byserum from these refractory patients, and the ag-glutination was not inhibited by prior absorptionof these sera with human gastric juice. It is ap-parent therefore that, although the phenomenonof acquired resistance to orally administered hogintrinsic factor may have an immunological basis,it is unrelated to the reaction of autoantibodieswith human intrinsic factor.

Further studies with intrinsic factor antibodymay not only facilitate the identification of intrinsicfactor in vitro, but may also extend our knowledgeof the physiologic and pathologic processes thatrelate to intrinsic factor secretion and vitaminB12 absorption.

SUMMARY

1. The interaction of sera from perniciousanemia patients and purified human intrinsic fac-tor (IF)-Co60B,2 complex has been studied by invivo absorption tests and by in vitro electrophoreticand immunologic methods.

2. Vitamin B12 absorption was inhibited whensome pernicious anemia sera were given orallywith IF-Co60B,2 to pernicious anemia patients.

3. Inhibitory pernicious anemia sera reactedwith IF-Co60B,2 in vitro. These sera altered theelectrophoretic mobility of IF-Co00B,2.

4. It was shown that -y-globulin from inhibitorysera combined with IF-Co6OB12: a) IF-Co60B,2migrated with the y-globulin fraction of inhibitorysera on paper electrophoresis; b) y-globulin pre-pared from inhibitory sera altered the electro-phoretic mobility of IF-Co6OB12 on starch gel.

5. A single radioactive zone of action between

inhibitory sera and IF-Co60B,2 was observed inagar diffusion studies.

6. The IF-Co60B12 binding by inhibitory perni-cious anemia sera was measured by electrophoresisof mixtures of sera with known concentrations ofIF-CoUMB12. The quantity of IF-Co60B12 boundby inhibitory sera was commensurate with the in-hibition of vitamin B12 absorption produced bythese sera in absorption tests.

7. It is concluded that antibody to human in-trinsic factor is present in some sera from patientshaving pernicious anemia. The significance ofthis intrinsic factor antibody in relation to theetiology of pernicious anemia is discussed.

ACKNOWLEDGMENTS

The cooperation of Dr. David V. Becker and the Radio-isotope Department of The New York Hospital in mak-ing available radioisotope equipment is gratefully acknowl-edged. The patients presented in this report were stud-ied through the courtesy of Dr. Ralph L. Engle, Jr. Wewould like to thank Dr. Henry Kunkel for helpful sug-gestions in the use of immunological techniques, Dr. Wil-liam Christensen and Dr. David V. Becker for criticalreview of the manuscript, and Mr. Lloyd Benjamin fortechnical assistance.

REFERENCES

1. Schwartz, M. Intrinsic-factor-inhibiting substancein serum of orally treated patients with perniciousanaemia. Lancet 1958, 2, 61.

2. Schwartz, M. Intrinsic factor antibody in serum frompatients with pernicious anaemia. Lancet 1960, 2,1263.

3. Taylor, K. B. Inhibition of intrinsic factor by per-nicious anaemia sera. Lancet 1959, 2, 106.

4. Taylor, K. B., and Morton, J. A. An antibody toCastle's intrinsic factor. J. Path. Bact. 1959, 77,117.

5. Lowenstein, L., Cooper, B. A., Brunton, L., andGartha, S. An immunologic basis for acquiredresistance to oral administration of hog intrinsicfactor and vitamin B,2 in pernicious anemia. J.clin. Invest. 1961, 40, 1656.

6. Kay, A. W. Effect of large doses of histamine ongastric secretion of HCl; an augmented histaminetest. Brit. med. J. 1953, 2, 77.

7. Jeffries, G. H., Hoskins, D. W., and Sleisenger, M.H. Determination of vitamin B,2 binding capacityof gastric juice by starch gel electrophoresis. Clin.Res. 1961, 9, 153.

8. Jeffries, G. H., Smith, F. W., Hoskins, D. W., andSleisenger, M. H. Fractionation of the non-dialys-able components of normal human gastric juice by

114

ANTIBODY TO INTRINSIC FACTORIN PERNICIOUS ANEMIA

starch gel electrophoresis. Gastroenterology 1961,41, 467.

9. Glass, G. B. Jerzy. Localization of intrinsic factorand the sites of B..-binding in the paper electro-phoresis of human gastric juices and concentratesfrom hog gastric mucosa. Haemat. latina 1959, 2,231.

10. Schilling, R. F. Intrinsic factor studies II. The ef-fect of gastric juice on the urinary excretion ofradioactivity after the oral administration of ra-dioactive vitamin B,.. J. Lab. clin. Med. 1953, 42,860.

11. Pert, J. H., Engle, R. E., Jr., Woods, K. R., andSleisenger, M. H. Preliminary studies on quan-titative zone electrophoresis in starch gel. J. Lab.clin. Med. 1959, 54, 572.

12. Berson, S. A., Yalow, R. S. Bauman, A., Rothschild,M. A., and Newerly, K. Insulin-I1'l metabolism inhuman subjects: Demonstration of insulin bindingglobulin in the circulation of insulin treated sub-jects. J. clin. Invest. 1956, 35, 170.

13. Unger, R. H., Eisentraut, A. M., McCall, M. S., andMadison, L. L. Glucagon antibodies and an im-munoassay for glucagon. J. clin. Invest. 1961, 40,1280.

14. Ouchterlony, 0. Antigen-antibody reactions in gels.IV. Types of reactions in coordinated systems ofdiffusion. Acta path. microbiol. scand. 1953, 32,231.

15. Pitney, W. R., Beard, M. F., and Van Loon, E. J.Observations on the bound form of vitamin B.2 inhuman serum. J. biol. Chem. 1954, 207, 143.

16. Mendelsohn, R. S., Watkin, D. M., Horbett, A. P.,and Fahey, J. L. Identification of the viamin B.!-binding protein in the serum of normals and ofpatients with chronic myelocytic leukemia. Blood1958, 13, 740.

17. Lear, A. A., Harris, J. W., Castle, W. B., and Flem-ing, E. M. The serum vitamin B,2 concentrationin pernicious anemia. J. Lab. clin. Med. 1954, 44,715.

18. Schwartz, M., Lous, P., and Meulengracht, E. Re-duced effect of heterologous intrinsic factor afterprolonged oral treatment in pernicious anaemia.Lancet 1957, 1, 751.

19. Hennes, A. R., Sevelius, H., Llewellyn, T., Woods,A. H., Joel, W., and Wolf, S. Experimental pro-duction of atrophic gastritis by a presumably im-munologic mechanism (abstract). J. Lab. clin.Med. 1960, 56, 826.

1115