Aspects of Vitamin B12 and Folate Metabolism in Malabsorption Syndromes

JOHN LINDENBAUM,

M.D.

New York. NW York

Recent clinical and physiologic observations suggest some modifications in traditional concepts of cobalamin absorption. Mild cobalamin deficiency may occur due to the inability to liberate the vitamin from food in certain patients who lack gastric acid and pepsin due to gastritis or following partial gastrectomy. Severe deficiency in these disorders will usually only develop if intrinsic factor secretion is compromised. Although proteolytic digestion of R binders by pancreatic enzymes is required for the Schilling test to be normal, the physiologic necessity of this step is not fully established, since patients with pancreatic insufficiency do not appear to develop cobalamin deficiency. Cobalamin lack itself may cause transient impairment of absorption of the vitamin. The initial differentiation of tropical sprue and pernicious anemia may be difficult, since both disorders often present with isolated cobalamin lack, with or without diarrhea. Routine use of assay of serum antibodies to intrinsic factor is a significant advance in the rapid diagnosis of pernicious anemia. Dietary folate polyglutamates require hydrolysis, probably by a small intestinal brush border enzyme, before monoglutamate absorption can occur. Folate malabsorption as the primary cause of megaloblastic anemia is uncommon, and is mainly seen in patients with sprue. In tropical sprue, secondary folate deficiency often appears to compromise gut function and precipitate symptomatic relapse. Vitamin BIz and folate deficiency are often major clinical developments in patients with malabsorption. The derangements in the normal physiology of the assimilation of these vitamins that occur in disease have been the subject of intensive study for several decades. This review will focus on our current knowledge of this area, with an emphasis on clinically important disturbances, such as the sprue syndromes. The effects of bacteria, parasites, drugs and ethanol on vitamin BIZ and folate absorption are discussed elsewhere by other contributors to this symposium.

VITAMIN Blz ABSORPTION

From the Medical Services, Harlem Hospital Center and Columbia-Presbyterian Medical Center, and the Department of Medicine, Columbia University, College of Physicians and Surgeons, New York, New York. Requests for reprints should be addressed to Dr. John Lindenbaum. Medical Service, Harlem Hospital Center, New York, New York 10037.

Vitamin BIz (cobalamin) is found almost exclusively in foods containing animal protein [l], although certain seaweeds [a] and well water contaminated with enteric bacteria [3] may serve as unusual sources of cobalamin. Because of the extensive storage of cobalamins in the body, strict vegetarianism for prolonged periods of time is required for the development of true nutritional vitamin BI:! deficiency in an adult. Therefore, in the United States at least, in almost all patients in whom significant cobalamin deficiency develops, a disturbance in the pathways for absorption of the vitamin can be demonstrated. Role of the Stomach. The various coenzyme forms of vitamin B12 present in foods [4] are bound to protein, although the exact nature of

December 1979

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VITAMIN

B12 AND FOLATE METABOLISM

TABLE I

Vitamin Blz Absorption from Eggs and Crystalline Cyanocobalamin” MeanPer Cent57C~alli24 hourUrine Crystalline Cyanocobalamln 899s

No. of Patients Normal controls Pernicious anemia Partial gastrectomy Hypolachlorhydria l

Based

IN MALABSORPTION

11

3.37

5 10 5

0.03 0.30 0.42

on data of Doscherholmen

18.1 0.8 17.5 26.7

and Swaim [lo].

the binding proteins remains to be elucidated. In order for food cobalamins to be absorbed, these bonds must be broken and the cobalamin ultimately transferred to intrinsic factor in the gastrointestinal lumen. Although published in vitro studies have been conflicting, most observers have found that a certain fraction of food vitamin B1sis liberated into a dialyzable form during the process of homogenization or cooking at neutral pH, with further generation of free vitamin B~zat low pH, with or without added pepsin [5-B]. The relative importance of cooking, acid and pepsin remains to be established, and probably varies with the individual food [5-81.

In recent years, a number of investigators have called attention to a subtle form of vitamin BQ malabsorption, in which cobalamin deficiency is believed to have developed due to inability to liberate cobalamins from food in the presence of adequate amounts of intrinsic factor. This may occur in patients who have had a partial gastrectomy or a vagotomy with gastroenterostomy, or who have chronic gastritis with achlorhydria or hypochlorhydria [g-16]. In most instances, patients have been noted to have low serum vitamin B12 concentrations despite the presence of a normal Schilling test performed with crystalline cyanocobalamin. When these patients were given radiolabeled cobalamin incorporated into eggs (Table I) or chicken meat, or crystalline cobalamin bound in vitro to ovalbumin or chicken serum, absorption of these protein-bound cobalamin preparations was subnormal. These observations appear to establish an entity of food-cobalamin malabsorption,

TABLE II

probably due to lack of acid and/or pepsin. It should be noted, however, that most of these patients have decreased, although not markedly diminished, intrinsic factor secretion, and crystalline cobalamin absorption by standard Schilling tests may be intermittently subnormal [17]. Furthermore, although low serum vitamin B12levels are not unusual in these patients, the development of frank vitamin B1zdeficiency is relatively rare, and when it occurs is often associated with other factors causing a negative balance of cobalamins, such as poor dietary intake or the presence of Diphyllobothrium latum infestation [15,18].These clinical observations are consistent with the in vitro findings of several workers indicating that a certain fraction of food vitamin Blz becomes dialyzable even without the addition of acid or pepsin [6,8].Thus, it is likely that any requirement for acid-peptic digestion of food cobalamin-protein bonds is only partial and will apply only to a fraction of the dietary vitamin sources. The requirement for intrinsic factor is absolute and is sensitively assayed by the routine Schilling test. In our experience, as well as that of others [19,20], virtually all patients in whom clinically significant vitamin Blz deficiency develops related to atrophic gastritis or partial gastrectomy will have abnormal absorption of crystalline cyanocobalamin. Once cobalamins are liberated from food, they must become bound (sooner or later] to intrinsic factor (Table II) in order for ileal uptake and absorption to occur. Intrinsic factor deficiency occurs most frequently as a result of severe chronic atrophic gastritis (adult-type pernicious anemia) or following partial or total gastrectomy (19,261.In patients with pernicious anemia the main cause of cobalamin depletion is lack of intrinsic factor, although binding of intrinsic factor by intraluminal antibodies, bacterial overgrowth secondary to achlorhydria and reversible ileal dysfunction due to vitamin B12deficiency itself may contribute to malabsorption of the vitamin [21,22]. Rarely, lack of vitamin Blz occurs early in childhood in patients with an apparent isolated inherited defect in intrinsic factor synthesis, in association with an otherwise normal stomach which secretes abundant hydrochloric acid (so called “congenital pernicious anemia”] [23]. Of great theoretic interest was the recent report of a child in whom co-

Human Vitamin B,,-Binding Proteins*

Protein

Origin

Intrinsic factor

Parietal cell

Transcobalamin II

Liver, ? gut, macrophages Many cells (including granulocytes)

R binders (including Transcobalamins I and Ill) l

SYNDROME-LINDENBAUM

Fluid Gastric juice Serum Serum, bile, milk, saliva, gastric juice

Molecular Weight

Glycoprotein

38,000

Polypeptide

58,00066,000

Glycoprotein

Adapted from Allen [25].

1038

December 1979

The American Journal of Medicine

Nature

44.000

Volume 97

Function Deliver dietary vitamin B12 to ileum Transport vitamin B12 from gut, deliver vitamin B12 to cells Transport vitamin Bj2 and analogues to liver, ? rid body of analogues. ? antibacterial

VITAMIN

B,3 AND

FOLATE

balamin deficiency developed due to secretion of an abnormal intrinsic factor that was able to bind vitamin B~z well but had markedly decreased ability to promote cobalamin uptake by ileal homogenates [%I. Such a disorder has not yet been described on an acquired basis. Role of the Pancreas. As a result of the elegant studies of Allen and co-workers [25,26], it is now realized that substantial amounts of dietary cobalamins may be bound in the gastrointestinal lumen, at least transiently, to so-called R binders [Table II) present in saliva, gastric juice, bile and intestinal juices. At low pH, R binders have a much greater affinity for cobalamins than intrinsic factor. and virtually all vitamin Blz added to an equimolar mixture of the two binders in purified form at pH 2 will be bound to the R binder [26]. Even at pH 8, two to three times as much cobalamin will be bound to the R binder (Table III). Since R binders do not promote ileal uptake of the vitamin, if gastrointestinal fluids were no more than a simplified mixture of these binders and dietary cobalamins, most of the vitamin Blz in an oral dose would not be absorbed [which is not the case in vivo]. Allen and co-workers [26] have shown that pancreatic proteases partially degrade R binders in vitro and allow vitamin Bj:! to be transferred with time to intrinsic factor. That a similar situation may occur in the gastrointestinal tract can be argued from the finding that approximately 50 per cent of the patients with pancreatic insufficiency absorb crystalline cobalamin poorly, and that this malabsorption may be completely corrected by the administration of pancreatic enzyme preparations or purified trypsin and partially corrected by the administration of bicarbonate [27-301. Further, the ingestion of a vitamin Bj2 analogue in amounts large enough to saturate gastrointestinal R binders normalized crystalline cobalamin absorption in patients with pancreatic insufficiency [31]. Other observations, however, call into question the absolute requirement for pancreatic proteases in cobalamin absorption in human subjects. Some patients who have undergone total pancreatectomy may have normal crystalline cobalamin absorption (321. In addition, in some patients with pancreatic insufficiency and abnormal Schilling tests, crystalline cobalamin absorption is normalized when radioactive vitamin Blz is given with a meal [29,33]. Finally, and most persuasively, cobalamin deficiency is extremely unusual (or does not occur) in patients with pancreatic insufficiency in the absence of other disturbances in cobalamin absorption, such as pernicious anemia. These observations suggest that even in the absence of pancreatic enzymes, enough dietary cobalamin is transferred to intrinsic factor to prevent the development of depletion of body stores. Studies in vivo of the actual luminal events in the processing of food cobalamins will be required to clarify these questions. In an in vivo study using an oral dose of crystalline cobalamin given in the fasting state to

METABOLISM

IN MALABSORPTION

SYNDROME--

LINDENBAUM

Distribution of Cobalamin between R Protein and Intrinsic Factor*

TABLE III

Per Cent VitaminB12 Boundto R Protein IntrinsicFactor 98 70

2 30

PH 2 PH 8

NOTE: Equimolar amounts of purified intrinsic factor and R protein were incubated with radioactive cyanocobalamin in buffered solutions for 60 minutes at 37%. Based on Allen et al. [26]. l

normal subjects as well as to patients with tropical sprue, 37 to 75 per cent of the radioactive vitamin Blz was found to be bound to intrinsic factor in the first loop of jejunum [34]. Ileal Events. Cobalamins are present in high concentration in bile, probably bound to R binders [25]. Although it has often been assumed that there is an extensive enterohepatic circulation, the percentage of biliary vitamin Blz that is reabsorbed in the ileum is uncertain [35]. Since the ileum is the main site of active vitamin BiZ absorption from the vitamin concentrations present in food (although passive absorption of cobalamins given in massive doses may occur throughout the small bowel], any disorder involving the ileum may lead to deficiency. Understanding of the events occurring in the ileum during cobalamin transport into the portal venous system remains limited. The presence of a microvillus re-

L

:

I

cy o;y

d

_-

P---P

2

345678

I

I

I

I

1

I

9

to

II

SEGMENT NUMBER sy_-__+op 3~~___________..---ss~

DUODENUM

JEJUNUM

T-

Figure 1. Bindina of 57Co-B13 bound to ourified intrinsic factor by homogenates of 60 dk segments of human small bowel obtained at autopsy from three subjects with no intestinal disease. Bars indicate standard errors of the means. In each specimen 93 to 98 per cent of all binding was localized to segments 6 to 11 (ileum). From Hagedorn and Alpers

December 1979

[371.

The American Journal of Medicine Volume 67

1039

VITAMIN

B12 AND FOLATE METABOLISM

25~

m

.

I

I

1

IN MALABSORPTION

.

. 20-

: l*

E

.

LENGTH

cm

I

:igure 2. Correlation (r = 0.76) between urinary excretion of radioactive vitamin B12 (given without intrinsic factor) in the Schilling test and the length of ileum resected as measured at the time of surgery in 136 patients studied by Filipsson et al. [47].

ceptor site for the vitamin B1z-intrinsic factor complex has been well demonstrated, as well as the pH and calcium dependence of this step, which involves a nonenergy-requiring attachment to a membrane protein that has recently been solubilized but not purified [19,36].These receptor sites have been found throughout the distal three/fifths of the human small bowel (Figure l), with maximal activity just before the terminal ileum [37]. A delay of several hours occurs during the uphill transport of cobalamin across the ileal cell which occurs against a concentration gradient (as in the Schilling test]. In animals, vitamin B1z is transiently associated with mitochondria during this delay period [38]. Cobalamin emerges from the ileal cell and is bound to transcobalamin II in the portal circulation; intrinsic factor is not absorbed [19,25,39]. Whether the presence of transco-

TABLE IV

Cause

SYNDROME-LINDENBAUM

balamin II in the circulation (or, indeed, its synthesis by the gut cell) [39] is required in order for absorption to occur is uncertain. Of the three families reported on with congenital transcobalamin II deficiency, three members have been studied and all were found to malabsorb vitamin B12[40-421. Rigorous proof that this binder is essential for cobalamin absorption however, is lacking. Which of the several ileal steps in vitamin Blz assimilation is interrupted by various disease states is obscure. In the single patient with hereditary selective vitamin Blz malabsorption that has been studied to date, the binding activity of the iIea1 receptor site for the vitamin Blz-intrinsic factor complex was found to be normal [43]. The relatively common disorders causing clinically significant vitamin Blz deficiency due to ileal dysfunction include ileal resection, regional enteritis, tropical sprue and celiac disease [19]. The occurrence of megaloblastic anemia and abnormal tests of crystalline cobalamin absorption following ileal resection can generally be correlated with the amount of distal bowel removed (Figure 2) [19,44-471. Patients with less than 50 to 55 cm of resected ileum are unlikely to have vitamin B1zmalabsorption [19,44-471.Similarly, in patients with regional enteritis, the extent of diseased ileum as well as previous surgical resection, appear to determine the likelihood of cobalamin malabsorption [44,46,47]. In patients who have had jejunoileal bypass procedures for obesity, the incidence .of crystalline cobalamin malabsorption has varied in several published series [48-521. Multiple factors, including the length,of ileum remaining in continuity, reflux of vitamin Blx into bypassed ileum and bacterial overgrowth, undoubtedly play a role. Whether adaptive improvement in cobalamin absorption may occur with time in such patients is uncertain [48,50-521. Megaloblastic anemia is extremely common in patients with tropical sprue, and may be due to lack of folate, cobalamin or both (Table IV). In some patients, isolated vitamin Blz deficiency may occur [Table IV) [53]. Possibly, they have been protected from folate deficiency as the result of vitamin synthesis by jejunal bacteria [54]. Although a minority of patients have an

of Megaloblastic Anemia in 63 Patients with Tropical Sprue 1960-1976

SerumFolate

SerumVitamin8,s

1960-1967’

1966-1976t

NO.

%

Normal Low Low Low

2 25 5 3

2 7 9 10

4 32 14 13

6.3 50.8 22.2 20.6

35

28

63

Low Low Borderline Normal or high Total patients NOTE: Patients with biopsydocumented 1960-1978. From Klipstein and Falaiye [53]. 7 Lindenbaum J: Unpublished data.

tropical sprue presenting at Columbia University-affiliated hospitals in New York City during

l

1040

December

1979 The American Journal of Medicine

Volume 67

VITAMIN

B,:: AND

FOLATE

METABOLISM

IN hlALABSORPTION

SYNDRORIE

m-I.INDENBAIJM

16. 14-

. 2

.

5 4. ii

l

i ‘t OL

. .

.

XYLOSE

FAT

igure 3. Intestinal absorption of xylose, vitamin B12 and fat in 28 untreated patients with cobalamin deficiency due to pernicious anemia. In studies of vitamin 812 absorption, intrinsic factor was given with the radioactive cobalamin in the Schilling test [22].

gastritis severe enough to impair intrinsic factor secretion, in most the disturbance in vitamin BU absorption appears to be primarily ileal [34,55]. However in some series, in about half the patients cobalamin malabsorption was rapidly corrected by antibiotic therapy, suggesting that the vitamin had been bound by intraluminal bacteria [56,57]. In patients with celiac disease, clinically significant vitamin B1z deficiency is distinctly unusual, reflecting the lesser degree of ileal injury in the average patient [58]. The number of patients who have abnormal crystalline cobalamin absorption, however, has varied from 18 per cent to more than 50 per cent in various published series [49.58,59]. In a small group of patients in whom ileal biopsy specimens were obtained, there was a good correlation between the height of ileal epithelial surface cells and cyanocobalamin absorption [58]. Cobalamin deficiency itself may cause a morphologically “megaloblastic” gut which reverts to normal after vitamin therapy [60]. In three quarters of the patients with pernicious anemia, the deficiency state was associated with malabsorption of crystalline cobalamin given with intrinsic factor (Figure 3) [z]. Intrinsic factor-mediated cobalamin absorption usually returned to normal within one to eight weeks of treatment with the vitamin (Figure 4) [22]. Therapy with a number of drugs [discussed elsewhere in this symposium), including para-amino salicylic acid,

associated

neomycin, colchicine and biguanides, as well as chronic intoxication with alcohol, may cause cobalamin malabsorption, presumably at the ileal level. However, there are no convincing reports of the development of clinically significant deficiency as a result of the ingestion of any of these agents. Massive hypersecretion of acid caused by a gastrinproducing tumor (the Zollinger-Ellison syndrome) may be associated with vitamin BQ malabsorption which is correctable by bicarbonate administration. The exact mechanism is unknown [61], but possible explanations could invoke impairment of the pH-dependent uptake by ileal microvillus receptors, or increased association of cobalamins with R binders at lowered intestinal PH. When the physProblems in Differential Diagnosis. iology of cobalamin absorption is viewed from a clinical standpoint, it is apparent that the absolute requirements for vitamin Blz absorption from food are the presence of intrinsic factor and an intact ileum. The Schilling test remains the cornerstone of diagnosis in most patients in whom clinically significant vitamin B12 deficiency develops. In such patients, an abnormal Schilling test is the rule. In a recent series of 123 consecutive patients with megaloblastic anemia due to cobalamin lack (Table V), crystalline cobalamin absorption was normal in only two [patients with multiple jejunal diverticulas who had recently received antibiotics for other disorders). In this

December 1979

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1041

VITAMIN

B,Z AND

FOLATE

METABOLISM

0

IN MALABSORPTION

7

14

21

DAYS

t

Figure 4. dose given

26

Improvement

in urinary

35

of vitamin

_ Cause Pernicious anemia* Partial or total gastrectomy Multiple jejunal diverticula Tropical Spruet Regional enteritis +/ileal resection Unknown cause (workup incomplete)

a7

70.7

4 5 21 4 2

3.3 4.1 17.1 3.3 1.6

NOTE: Patients admitted to Harlem or Presbyterian Hospitals, 1967-1977. Includes one patient who also had WaldenstrGm’s macroglobulinemia with ileal involvement. + Includes seven patients with combined vitamin Bq2 and folate deficiency. l

1979

56

57Co-cyanocobalamin

Cause of Cobalamin Deficiency in 123 Consecutive Cases of Megaloblastic Anemia in New York City

December

49

71

99

I46

OF B,,THERAPY

series, one (or more] of three clinically important causes of cobalamin deficiency was present: lack of intrinsic factor, bacterial overgrowth or ileal disease (Table VI. Although an accurate history often provides crucial clues, an etiologic diagnosis cannot be made with certainty without laboratory support. Physicians often persist in the erroneous belief that the absence of diarrhea argues strongly against an underlying malabsorption syndrome. At least 10 per cent of patients with tropical sprue who present with a megaloblastic anemia, for example, have no gastrointestinal symptoms [53], and the majority of vitamin Bi2-deficient persons with multiple jejunal diverticulas give no hint by history of an enteric disorder. Conversely, as many as a quarter

1042

42

excretion

after an ori

with intrinsic factor during vitamin therapy of 10 patients with pernicious

anemia in whom malabsorotion

TABLE V

SYNDROME-LINDENBALJM

The American Journal of Medicine

B12 was present

in the untreated

state

to a third of patients with uncomplicated pernicious anemia complain of diarrhea [22,62], although constipation is even more common. The transient.small bowel dysfunction that results from vitamin Blz deficiency [22,63] may cause diagnostic problems. Since ileal and jejunal function return to normal within seven days of cobalamin therapy in most patients [Figure 41, we usually defer Schilling or xylose testing until one week of vitamin Blz treatment has been given. In the minority of patients with pernicious anemia in whom crystalline cobalamin absorption with added intrinsic factor remains subnormal for longer periods, or in those in whom an accurate Schilling test cannot be performed (due tb renal failure or inability to collect a 24 hour urine), two tests are useful. An assay for serum antibodies to intrinsic factor, a simply performed procedure [64a] that will be positive in approximately two thirds of the patients with pernicious anemia, will often provide a rapid diagnosis. Finally, analysis of gastric acid secretion after pentagastrin or betazole stimulation is no longer necessary or indicated when the diagnosis can be made by Schilling tests or if the antibody assay is positive. Gastric analysis may provide useful indirect information, however, when the latter tests do not clearly point to the cause of cobalamin deficiency. FOLIC

ACID ABSORPTION

Malabsorption of folic acid is a much less frequent cause of clinically important lack of the vitamin than is the case with cobalamins. The great majority of patients with megaloblastic anemia due to lack of the vitamin in

Volume 67

VITAMIN

B,:: AND

FOLATE

METABOLISM

IN MALABSORPTION

SYNDROhlE-I,INI)F.NBAl~Jhl

14C-PG-I

“C-PG-7

CM

FROM INFUSION

PORT

i folates appearing as heptaglutamate (PG-7, Figure 5. Percentage of T-labeled left) and monoglutamate (PG-1, right) during 56 minutes of perfusion of 50 cm segments of upper jejunum of five normal volunteer subjects with 14C-labeled heptaglutamate. Increasing amounts of labeled monoglutamates (as determined by column chromatography) recovered distal to the infusion port reflect progressive hydrolysis of the infused polyglutamate. From Halsted et al. [74].

the United States are chronic alcoholics who have been eating poorly [64b]. Malabsorption does not appear to be the primary cause of folate depletion in this group [65]. Nonetheless, folate deficiency due to impaired absorption is likely to occur in certain patients, especially those with extensive mucosal disease of the duodenum and jejunum. Folates in Food. The absorption of dietary folate has been the subject of recent review articles [64b,66,67]. Very little pteroylglutamic acid or other oxidized folates are present in foods [67,68]. In almost all of the dietary folate, the double bonds on the pyrazine ring of the pteridine moiety of folic acid are reduced, and a methyl or formyl group is attached at the Ns or NIO position [67-691. In the majority of foods that have been well characterized, such as lettuce, cabbage, liver or orange juice, most of the folate is present as polyglutamates with five or more glutamate residues added in gamma-carboxy1 linkage to the single glutamic acid found in pteroylmonoglutamate [67]. Very little reliable data are available concerning the availability of folate from different items of the diet under experimental conditions resembling the real-life intake of foods. The best currently available methods require continuous high level saturation with dietary folate and the ingestion of huge amounts of the foods under study in order to obtain semireproducible values for urinary folate excretion.

December

Under these conditions, the availability of folate from various food items has varied widely [70-721. Hydrolysis of Polyglutamate Folates. With the recent development of biochemical techniques for the solidor liquid-phase synthesis of radiolabeled polyglutamates, important information has been generated concerning the mechanism of absorption of folates in human subjects [66,73]. Purified polyglutamates have been shown to be substantially absorbed in human subjects, although less efficiently than monoglutamates [66,74,75]. In jejunal perfusion studies in normal subjects and in patients with sprue syndromes, the luminal disappearance of polyglutamate folates was less rapid than that of equimolar amounts of monoglutamates, suggesting a limiting effect of hydrolysis on absorption [74-761. Hydrolysis of polyglutamates to monoglutamates appears to be an obligatory step in the absorption of folates from polyglutamates. When the human small intestine is perfused with polyglutamate folates, hydrolysis products with decreasing number of glutamate residues down to the monoglutamate form (Figure 5) rapidly appear in the gut lumen distal to the site of perfusion, suggesting that such hydrolysis goes on at the cell surface. Such an interpretation would require the presence of an active hydrolytic enzyme (called by various investigators “conjugase,” “pteroyl polyglutamyl hydrolase,” or

1979

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VITAMIN

B12 AND FOLATF METABOLISM IN MALABSORPTION

“gamma-glutamyl-carboxy-peptidase”) on the cell surface. Most workers, however, have reported that enzyme activity in intestinal cells is largely localized to lysosomal fractions [77,78]. This dilemma appears to have been resolved recently by the report of Reisenauer and colleagues [79] of two separate enzyme activities in human jejunum, one intracellular with a pH optimum of 4.5 (presumably lysosomal) and the other localized to brush border membranes, with a pH optimum of 7.5. Monoglutamate Absorption. Monoglutamate folates present in the diet as well as those generated by intestinal cell hydrolysis of polyglutamates appear to share a common final. pathway, that of monoglutamate transport across the cell [66]. Considerable conflicting evidence from many laboratories has accumulated from in vitro and in vivo studies in various species concerning the mechanism of monoglutamate transport [66]. In man, for example, evidence for a saturable mechanism and for transport against a concentration gradient has been obtained from perfusion studies [80]. On the other hand, the total amounts of folate absorbed from tritiated monoglutamates appear to be similar over a dose range of 25 to 3,000 pg [62]. The most convincing argument for the existence of a specific transport mechanism is the occurrence of rare cases of congenital isolated folate malabsorptibn in the absence of other disturbances of intestinal function [81,82]. Such patients have been shown to have markedly impaired absorption of polyglutamate and monoglutamate folates (including oxidized, reduced, methylated and formylated forms] and require large doses of oral folic acid to prevent recurrence of megaloblastic anemia. The four patients described have all had varying degrees of mental retardation, and transport of folates into the central nervous system also appears to be abnormal [81]. The controversy regarding the manner in which monoglutamate folates are transported may be partly a reflection of differing experimental conditions [66] but may best be resolved by postulating the existence of a dual mechanism, i.e., a carrier-mediated, saturable, uphill transport system for low concentrations, and passive transport at higher concentrations. There is increasing evidence for the presence of such a dual system in animals [83-851. Monoglutamate folate absorption in man is depressed by alkalinization of the jejunum [84,86,87]. Although it has been proposed that this is related to increased ionization of folic acid above the pKa’s of its glutamyl carboxy1 groups, resulting in decreased passive diffusion of the nonionic form of the molecule [88], in rat jejunum it is the saturable component of folate upttike that is pH-sensitive [84]. Alteration of the folic acid molecule (e.g., reduction and methylation) by the intestinal cell may occur during absorption, particularly at low concentrations, but is not obligatory for folate transport [89-921. Folates are excreted in high concentration into bile (mainly in monoglutamate forms) and are reabsorbed by the small intestine [93].

1044

December 1979

The American Journal of Medicine

SYNDROME-LINDENBAUM

Sprue Syndromes. Folate deficiency is encountered clinically as a primary result of impaired intestinal absorption most frequently in patients with marked villous atrophy of the upper small intestine. In celiac sprue (gluten-induced enteropathy) folate malabsorption is the rule, and megaloblastic anemia is common. Depressed serum or red cell folate levels have been found to be a useful screening test for the disorder and may be a sensitive indicator of noncompliance with a glutenfree diet [94-971. In patients with celiac sprue, absorption of both polyglutamate and monoglutamate folates is impaired [75,98-1001.In jejunal perfusion studies, the rate of hydrolysis of polyglutamates was estimated to be depressed, although the main disturbance appears to be in the uptake of monoglutamates [75,100]. A report that the pH of the lumen of the jejunum is increased in patients with celiac disease [IOI] has not been confirmed by others [87]. In a subgroup of patients with celiac disease, the surface pH of jejunal biopsy specimens (as measured in vitro) was found to be abnormally high [102]. Although loss of villus cells is a likely cause of folate malabsorption in this disorder, an increase in pH at the normally acid “microclimate” of the brush border could contribute to monoglutamate malabsorption in some patients [88,102]. Folate deficiency is a frequent cause of megaloblastic anemia in patients with tropical sprue (Table IV). Jejunal perfusion studies have demonstrated impaired uptake of folate from purified monoglutamates [76,103,104] and polyglutamates [76]. The degree of abnormality of monoglutamate uptake has been variable and in some cases was only demonstrable in the presence of glucose in the perfusing solution [103,104]. In some patients with tropical sprue in whom folate deficiency was present, absorption of crystalline monoglutamate was found to be normal [105-1071. Deficiency may develop on a diet rich in folates and respond hematologically to tiny doses of oral folic acid [108]. In addition, in some patients with normal absorption of crystalline monoglutamate, malabsorption of a yeast polyglutamate preparation has been found [107]. These observations suggest that a subgroup of patients with tropical sprue may be able to absorb purified monoglutamates but are unable to process food folates. The possible importance of conjugase inhibitors, such as pulses [log], folate-adsorbing substances [IIO] or folate-binding proteins in the diet of patients with the disease requires further exploration. The cause of tropical sprue is unknown. Three factors may play important roles in its pathogenesis [56]: (I] Secondary effects of folate and/or vitamin B12 deficiency on the gut. Not only is anemia corrected by vitamin therapy, but also many patients respond with cessation of intestinal symptoms and improvement in absorptive function and jejunal morphology [53,56,111]. (2) Bacterial overgrowth. Most workers now agree that jejunal colonization by coliform bacteria can be demonstrated in almost all patients with tropical sprue

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\‘ITAbIIN B,A AND FOLATE METABOLISM IN MALABSORPTION SYNDROIIE

[ll?,113]. These organisms secrete enterotoxins [ll3] (and possibly make other substances such as ethanol) which may be responsible for abnormalities in intestinal function (such as the secretion of salt and water) and mucosal morphology. (3) An unknown primary or initiuting factor. This would produce bowel injury, interfering with the clearance of coliform bacteria or accelerating the development of folate and vitamin B1z deficiency [56;112]. Other Intestinal Disorders. In a number of other conditions in which folate deficiency may occur, folate malabsorption may be one of several factors contributing to negative vitamin balance, but not necessarily the most important one: or the contribution of malabsorption may vary from patient to patient. In regional enteritis, for example, hematologically mild to moderate folate deficiency is common [ll4,115]. Inadequate dietary intake may be the most important cause [114]. In snme patients, however, especially those with duodenal and jejunal involvement by the disease, folate malabsorption may be significant [114,116]. Therapy of the disorder with sulfasalazine may also impair folate assimilation [ 1151. Since folio: acid appears to be better absorbed in humans from the upper small bowel than from the ileum, surgical resection of the duodenum and jejunum should impair folste absorption in the absence of compensatory adaptation in the ileum. That the latter occurs for folate compounds is possible [loo] but has not been established. Folate absorption has been found to be depressed in some but not all patients with jejunal resection [ll7-1211. Deficiency of the vitamin has not been a common complication in this group of patients (perhaps a reflection of their rarity plus the use of prophy-

LINDENBAUhl

lactic vitamins postoperatively]. When deficiency has been encountered. other factors [such as poor dietary intake, concomitant ileal resection, or very rapid intestinal transit) may have contributed to negative folate balance [IX?]. In a number of other conditions associated with small bowel dysfunction, abnormal folic acid absorption tests have been reported in certain patients, but anemia due to folate deficiency has not been encountered clinically. These include scleroderma [118], amyloidosis [118], diabetic enteropathy [118], congestive heart failure [123], Whipplc’s disease [118], partial gastrectomy [124], and hypoparathyroidism [318]. Folate malabsorption may also occur in patients with lymphomas [118,125] and (rarely] with the blind loop syndrome [126], but it has yet to be convincingly demonstrated that deficiency occurs as a result of malabsorption in these settings. The administration of various drugs and toxins, including sulfasalazine, diphenylhydantoin and ethanol, may be associated with malabsorption of monoglutamate folate. This is discussed elsewhere in this symposium, as well as in recent reviews [64b,65,66]. Folate deficiency itself, presumably in relation to the development of “megaloblastic” changes in the small intestine [127], may be associated with transient jejunal dysfunction, e.g., xylose malabsorption [65,128]. Because of the strong association between alcoholism and folate deficiency, it has been difficult to disentangle the respective roles of ethanol intake and flolate lack on gut function [129]. which indeed may be synergistic [13&131]. In jejunal perfusion studies, the absorption of folic acid itself, at least in monoglutamate form, has not been shown to be impaired by folate deficiency in the absence of alcohol ingestion [128.?30].

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Adams IF. McEwan F. Wilson A: The vitamin B-7 content of mcais and items of diet. Br ] Nutr 29: 65, 197$: Ericson LE. Banhidi CG: Bacterial growth factors related to vitamin Blz and folinic acid in-some brown and red seaweeds. Acta Chem Stand 7: 167.1953. Baker SJ. quoted in Britt RP, Harper C, Spray GH: Megaloblastic anaemia among Indians in Britain. Q J Med 160: 511. 1971. Farquharson 1, Adams JF: The forms of vitamin B1zin foods. Br J Nutr 36: 127, 1976. Rcizenstein PG: Effect of digestive enzymes on bound vitamin Bl-. Acta Med Stand 165: 481. 1959. Cooper BA, Castle WB: Sequential mechanisms’in the enhanced absorption of vitamin Blz by intrinsic factor in the rat. J Clin Invest 39: 199. 1960. Schade SC, Schilling RF: Effect of pepsin on the absorption of food vitamin B,? and iron. Am 1 Clin Nutr 20: 636, 1967 Adams JF. Kennedy EH, Thompson J, et al.: The effect of acid peptic digestion on free and tissue-bound cobalamins. Br ) Nutr 22: 111, 1968. blahmud K. Ripley D, Doscherholmen A: Vitamin BXZabsorption tests. Their unreliability in postgastrectomy states. ]AhlA 216: 1167, 1971.

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Doscherholmen A, Swaim WR: Impaired assimilation of egg 57Co vitamin BIG in patients with hypochlorhydria and achlorhydria and after gastric resection. Gastroenterology 64: 913. 1973. Streeter AM, Duriappah B. Boyle R, et al.: Malabsorption of vitamin B12after vagotomy. Am 1Surg 128: 340. 1974. Doscherholmen A. McMahon J, Ripley D: Inhibitory effect of eggs on vitamin B12absorption: description of a simple ovalbumin 57Co-vitamin Bl? absorption test. Br J Haematol 33: 261. 1976. Streeter AM, Shum HY, Duncombc Vhl. et al.: Vitamin Bls malabsorption associated with a normal Schilling test result. Med J Aust 1: 54,1976. King C, Leibach J, Toskes P: Food-bound vitamin B12malahsorotion-a clinicallv imoortant cause of vitamin B,? ._ deficiency. Gastroenteiolo& 72: 1080. 1977. Carmel R: Nutritional vitamin-B13 deficiency. Possible contributory role of subtle vitamin BIZmalabsorption. Ann Intern Med 88: 647,1978. Doscherholmen A, McMahon 1. Ripley D: Vitamin B12assimilation from chicken meat. Am J Clin Nutr 31: 825, 1978.

17. Adams JF, Cartwright Ej: The reliabiliw and reproducibility of the Schilling test in primary malabsorptive disease and

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after partial gastrectomy. Gut 4: 32, 1963. 18. Salokannel J: Intrinsic factor in tapeworm anemia. Acta Med

Stand 189 (suppl517): 1,197l. Donaldson RH: Mechanisms of malabsorption of cobalamin. Cobalamin (Babior BM, ed], New York, John Wiley & Sons, 1975, p 335. 20. Hines JD, Hoffbrand AV, Mollin DL: The hematologic complications following partial gastrectomy. Am J Med 43: 55. 1967. 21. Rose MS, Chanarin I: Intrinsic factor antibody and absorption of vitamin Brs in pernicious anemia. Br Med J 1: 25,1971. 22. Lindenbaum J, Pezzimenti JF, Shea N: Small-intestinal function in vitamin B,z __deficiencv. Ann Intern Med 80: 326, 19.

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McIntvre RO. Sullivan LW, leffries GH. et al.: Pernicious anemia in childhood. N Engl J Med 272: 981.1965. Katz M, Lee SK, Cooper BA: Vitamin Blz malabsorption due to a biologically inert intrinsic factor. N Engl J Med 287: 425, 1972. Allen RH: Human vitamin Brz transport proteins. Progr Hematol9: 57.1975. Allen RH, Seetharam B, Podell E, et al.: Effect of proteolytic enzymes on the binding of cobalamin to R protein and intrinsic factor. In vitro evidence that a failure to partially degrade R protein is responsible for cobalamin malabsorption in pancreatic insufficiency. J Clin Invest 61: 47, 1978. Veeger W. Abels J, Hellemans N, et al.: Effect of sodium bicarbonate and nancreatin on the absorption of vitamin B1s and fat in pancreatic insufficiency. NEngl J Med 267: 1341,1962. Toskes PP, Hansel1 J, Cerda J. et al.: Vitamin Brz malabsorption in chronic pancreatic insuficiency. Studies suggesting the presence of a pancreatic “intrinsic factor.” N Engl J Med 284: 627,197l. Matuchansky C, Rambaud JC, Modigliani R, et al.: Vitamin Brs malabsorption in chronic pancreatitis. Gastroenterology 67: 406, 1974. Toskes PP, Deren JJ, Conrad ME: Trypsin-like nature of the pancreatic factor that corrects vitamin Blz malabsorption associated with pancreatic dysfunction. J Clin Invest 52: 1660,1973. Allen RH. Seetharam B. Allen NC. et al.: Correction of cobalaminmalabsorption in pancreatic insufficiency with a cobalamin analonue that binds with high affinitv to R protein but not to intrinsic factor. In vivo evidence”that a failure to partially degrade R protein is responsible for cobalamin malabsorption in pancreatic insufficiency. J Clin Invest 62: 1628,1978. McIntyre PA, Sachs MV, Krevans JR, et al.: Pathogenesis and treatment of macrocytic anemia. Information obtained with radioactive vitamin Blz. Arch Intern Med 98: 541, 1956. Henderson JT, Warwick RRG, Simpson JD, et al.: Does malabsorotion of vitamin B,p occur in chronic oancreatitis? Lancet 2:‘241,1972. _Kaoadia CR. Bhat P. lacob E. et al.: Vitamin B17 absornt&n-a study of intraluminal events in control s&Jects and patients with tropical sprue. Gut 16: 98.1975. Grasbeck R, Nyberg W. Reizenstein P: Biliary and fecal vitamin Brs excretion in man. An isotope study. Proc Sot Exp Biol Med 97: 780.1958. Katz M, Cooper BA: Solubilized receptor for intrinsic factor-vitamin B,? comnlex from auinea Die intestinal mucosa. J Clin Invest 54: 733.1974. y LHanedorn CH. Alners DH: Distribution of intrinsic factorvitamin Brs receptors in human intestine. Gastroenterology 73: 1019,1977. Peters TJ. Hoffbrand AV: Absorption of vitamin Brz by the guinea-pig. I. Subcellular localization of vitamin Blz in the &al enterocyte during absorption. Br J HaematollS: 369, 1970. Chanarin I, Muir M, Hughes A, et al.: Evidence for intestinal origin of transcobalamin II during vitamin Brs absorption.

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Br Med 1 1: 1453.1978. Hakami N, Neiman PE, Canellos GP, et al.: Neonatal megaloblastic anemia due to inherited transcobalamin II deficiency in two siblings. N Engl J Med 285: 1163, 1971. 41. Hitzig WH, Dohmann U, Pluss HJ, et al.: Hereditary transcobalamin II deficiency: clinical findings in a new family. J Pediatr 85: 622.1974. 42. Burman JF, Mollin DL, Sladden RA, et al.: Inherited deficiency of transcobalamin II causing megaloblastic anemia. Br J Haemato135: 676,1977. 43. MacKenzie IL, Donaldson RM Jr, Trier JS, et al.: Ileal mucosa in familial selective vitamin Blz malabsorption. N Engl J Med 286: 1021,1972. 44. Gerson CD, Cohen N, Janowitz HD: Small intestinal absorptive function in regional enteritis. Gastroenterology 64: 907. 1973. 45. Fromm H. Thomas PI, Hofmann AF: Sensitivitv and specificity in tests of distal ileal function: prospective comparison of bile acid and vitamin Blz absorption in ileal resection patients. Gastroenterology 64: 1077,1973. 46. Lenz K: The effect of the site of lesion and extent of resection on duodenal bile acid concentration and vitamin Brz absorution in Crohn’s disease. Stand 1Gastroenterol 10:241.

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47. Filipsson S, Hulten L, Lindstedt G: Malabsorption of fat and vitamin Brz before and after intestinal resection for Crohn’s disease. Stand J Gastroenteroll3: 529,1978. 48. Nygaard K, Helsingen N, Rootwelt K: Adaptation of vitamin B17absorution after ileal bvoass. Stand 1Gastroenterol5: *. 349,197o: 49. Fausa 0: Vitamin BqTabsorotion in intestinal diseases. Stand J Gastroenterol 9 (&ppl29): 75,1974. 50. HiDDe E. luhl E, Bruusnaard A. et al.: Malabsorntion of vitamin I&i in obese paients treated with jejunoileal shunt. Stand J Gastroentero181 (suppl29) 1974. 51. Coyle JJ, Varco RL, Buchwald H: Vitamin Bls absorption followine human intestinal bvoass surgerv. Digestive “& _” Diseases-22: 12,1977. 52. Solhaug JH, Grundt I: Metabolic changes after jejuno-ileal bypass for obesity. Stand J Gastroent 13: 169.1978. 53. Klinstein FA. Falaive IM Tronical sorue in exoatriates from the tropics 1iving;n ‘the coniinental United ‘States. Medicine 48: 6, 1969. 54. Klipstein FA, Samloff IM: Folate synthesis by intestinal bacteria. Am J Clin Nutr 19: 237, 1966. 55. Wheby MS, Bayless TM: Intrinsic factor in tropical sprue. Blood 31: 6,1968. 56. Lindenbaum J: Tropical enteropathy. Gastroenterology 64: 637,1973. 57. Tomkins AM, Smith T, Wright SG: Assessment of early and delayed responses in vitamin Brs absorption during antibiotic therapy in tropical malabsorption. Clin Sci Mol Med 55: 533, 1978. 58. Stewart JS, Pollock DJ, Hoffbrand AV, et al.: A study of proximal and distal intestinal structure and absorptive function in idiopathic steatorrhoea. Q J Med 36: 425. 1967. 59. Fone Dl, Cooke WT, Mevnell Ml, et al: Co Brz absorption (hepatic surface count] after gastrectomy, ileal resection, and in coeliac disorders. Gut 2: 218, 1961. 60. Foroozan P, Trier JS: Mucosa of the small intestine in pernicious anemia. N Engl J Med 227: 553, 1967. 61. Schimoda S, Saunders DR, Rubin CE: The Zollinger-Ellison syndrome with steatorrhea. II: The mechanism of fat and vitamin B,s malabsorption. Gastroenterology 55: 705, 1968. 62. Chanarin I: The Meealoblastic Anaemias. Philadelohia. L Blackwell, 1969. ” 63. Mevers S. Schweitzer P. Gerson CD: Anemia and intestinal dysfunction in former residents of the Caribbean. Arch Intern Med 137: 181,1977. 64. (a) Gottlieb C, Lau KS, Wasserman LR, et al.: Rapid charcoal assay for intrinsic factor [IF), gastric juice unsaturated B12 binding capacity, antibody to IF, and serum unsaturated Blz binding capacity. Blood 25: 875,1965.

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VITAMIN BIZ AND FOLATE METABOI,ISM IN hlAI,ABSORPTION SYKDROE\IF, l,INT)ENIj.~1 lh4

[h) Lindcnbaum J: Malabsorption of vitamin Blz and folate. Current Concepts in Nutrition (Winick M, ed), ~017, New York. John Wiley & Sons, (in press]. 65. Baraona E. Lindenbaum J: Metabolic effects of alcohol on the intestine. Metabolic Aspects of Alcoholism (Lieher CS, ed). Lancaster, LtTP Press, 1977. p 81. 66. Rosenhcru IH: Absorntion and malahsorution of folatcs. Clin 67.

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Stnkstad ELR. Shin YS. Tamura T: Distribution of folate forms in food and folate availability. Folic Acid, Proceedinvs of ci Workshoo on Human Folate Reauirements. W&hington, Natinn’al Academy of Sciences, i977. p. 56. Perry J: Folatc analogucs in normal mixed diets. Br J Haern,ltol ‘ I“1: 435 1971. Butterworth C‘Elr. Santini R Ir. Frommever WB: The oteroylglutamntc components of American diets as determ’ined by chr,,matogr;lphic fractionation. J Clin Invest 42: 1929, 1!%3. Tamura ‘I’.Stnkstad ELR: The availahilitv of food folate in man. Br J Haomatol25: 513, 1973. ” Tamllra T. Shin YS. Beuhrine KU. et al.: The availahilitv of fnlates in man: effect of or&ge’juice supplement on”intcstinal conjugase. Br J Haematol 32: 123. 1976. Bsbu S. Srikantia SG: Availability of folates from some foods. Am J Clin Nutr 29: 376. 1976. Buttl:r\znrlh CE Jr, Baugh CM, Krumdieck C: A study of f~ll~itc:,Ibstrrptilln anti metabolism in man utilizing carbon-t4-labelt~d polyglutamates synthesized by the solid phase method. J Clin Invest 48: 1132, 1969. I-Ialstcd Cf I. Baugh CM. Butterworth CE Jr: Jejunal perfusinn 01 simnlc and coniugatcd fnlates in man. Gastroentclrology 68~261. 1975. ’ L’ HaIstctl CFI. Reisenauer AM Romero II. et at.: leiunal pcrfllsicm of simple and conjugated folates in celiad &rue. I Clin Invest 59: 933. 1977. C:‘orcinoJJ, Reiscnauer AM, Halsted CH: Jejunal perfusion trf simple and conjugated fnlates in tropical sprue. J Clin Invest 58: -~ “98 1976 Hoffl;rand AV. Peters TJ: The subcellular localization of ptcrnyl polyglutamatc hydrolase and folate in guinea pig intestinal mucosa. Biochim Bioohvs Acta 192: 479, 1969. Rosenberg IH, Godwin HA: The digestion and absorption of dietary folatc. Gestroenterology 60: 445, 1971. Reisenarler Ah4. Krumdieck CL, Halsted CH: Folate conjugase: two sc,paratr: activities in human jejunum. Science 198: 196. 1977. Hepner GW. Booth CC, Cowan J, ct al.: Absorption of crystalline folir: acid in man. Lancet 2: 302. 1968. I,;rnzko\vsky P: Congenital malabsorption of folate. Am 1 hll~ll 48: 580. 1970. Santi~lgo-Borrcn, PJ, Santini R Jr, Perez-Santiago E, et al.: Congenital isolatnd dcfcct of folic acid absorption. J Pediatr 8”: 450. 197,3. Halsted (XI. Bhanthumnavin K, Mezey E: Jejunal uptake of tritiated folic acid in the mt studied by in vivo perfusion. J Nulr 1114:1674. 1974. Russell RM. Jcelani Dhar G. Dutta SK, et al.: Influence of intraluminal pH on folatc absorption: studies in control subjects and in patients with pancreatic insufficiency. J Lab Clin 1 Mcd 93: 4”8 1079. R~,sc fiC. Koch K,li. i&hrwold DL: Folic acid transport by mamm;llian small intestine. Am J Physiol 235: E678. 1978. Benn A. Swan CIIJ, Cooke WT. et al.: Effect of intraluminal pf I cm the absorption of pteroylmonoglutamic acid. Br Med J 1: 148. 1971. MacKenzie JF. Russell RM: The effect of pH on folic acid ahsorption in man. Clin Sci Molec Med 51: 363, 1976. Blair IA. Mattv Al: Acid microclimate in intestinal absorotion.‘Ciin &tr&nterol3: 183. 1974. Baugh Ch4. Krumdieck CL, Baker HJ. et al.: Studies on the absorption and metabolism of folic acid. I. Folate absorption in the dog after exposure of isolated intestinal segments to synthetic pteroylpolyglutamates of various chain lengths. J Clin Invest 50: 2009. 1971.

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Whitehcad VM, Cooper BA: Absorption of unaltered folic acid from the gastro-intestinal tract in man. Br J IIacm,ltol 13: 679. 1967. Whitehead VM, Pratt R. Viallet A. et al.. Intestinal conversion of folinic acid to 5-methgltetrahyrlr(Ifl,I;lte in man. Br J Hacmatol 22: 63 1972. Leeming RI, Portn&-Graham H. Blair /A: The occurrence of folic~ac~d(I,terclyl-I,-monoglntamic a&l) in human hlood serum after small oral doses. J Clin Path 25: 491. 1972. Pratt RF, Cooper BA: Folates in plasma and bile of man after feeding folic acid-H and 5-formyltc:tr;~hydrofol;~ttl 1folinic acidl. 1Clin Invest 50: 455. 1971. hlnrtimcr PE. Stewart JS. Norman ,4P. et al.: f:ollow-up study of coeliac disease. Br bled J 3: 7. 1968. McNcish AS, Willoughby MLN: Whol+blood folate as a screening test for cocliac disease in childhond. I,anc:c:t 1: 44”

-I

1969:

Weir DG, Hourihane D O’B: Cneliac dise,lsc during the tcenagc period: the value of serial serum fnl,itc cstima&~ns. Gut 15: 450. 1974. 97. Hoffbrand AV: Anaemia in atltllt c~loliac cliscase. Clin Gastroentcrol 3: 71. 1974. 98. Rosenbert! IH. Godwin HA. Russell Rhl. et al.: Assessment of fnlatei;nalabsorption in deliac sprue com],;lring synthetic “H-1~ter~~ylpol~glutnmate. I”H-PteClu-] antI “-H-pteroylmtrnoglutomate (“H-PteGluJ. J Clin Invest 53: 66a. 1974. 99. Hoffbrand AV. Douglas AP. Fry I,, et al.: hl;llabsorption of dictury folatc ~ptero~lp0lyplllti~mates~ in adult coeliac disease and dcrmntitis hcroetiformis Br h,I~!tl I 4: 85.

96.

1970.

100. Halstcd (X. Rcisenauer AM. Shane B. et al.: Av,iilabilitv of nionnglutamyl and polyglutamyl folates in ncormal subiccts an11 in rraticnts with co&c snrm>. (;11t 19: 886. 1978. 101. Bonn A, Cooke W’T:Intraluminal pH oftlu~~denum and jcjunum in fasting subjects with normal and abnormal gastric or rlancreatic function. Stand I Gastro 6: 3 13. 1971. 10s LIIC~ h,lI.. Cooper BT, Lei FII, et al.: Acid microclimate in coeliac and Crohn’s discasr:: a modc!l for folatc malahsorption. Gut 19: 735, 1978. 103. Gersnn CD, Cohen N. Brnwn N, et al.: Fnlic ac:id and hcxose absorption in spruc. Am 1Dig Dis 19: 91 I, 1974. 104 Corcinb JJ. Colt i:, Klipstein FA: Pternylglutamic acid malabsorption in tropical spruc. Blood 45: 5;“;. 1075. 105. Paterson DE, David R. Baker SJ: Radiodiagnostic problems in malabsorption. Br [ Radio1 38: 181, 1!105. 106 Klipstein FA:&Ahsorption of physiologic doses of folic acid in suhjccts with tropical sprue r~:srlnncting !o tetracycline therapy. Blood 34: i9i. 19?9. . 107 floffbrand AV. Nccheles TF, Maldonatlo N. et al.: Malabsorntion nf fnlate nolvcrlutnmates in irnnical snrue. Br Mtrd . , J 2:‘543 1969. ’ .‘~’ 108 Shechv ?W. Rubini ME. Perez-Santiarro I?. et al.: The effect of “lninlltc” and “tit&ted” amountgof folic acid on the mcga~olrlastic anemia of tropical sprirc. f31ood 18: 623. 1961. 109 Bllttcrworth CE Tr. Newman Al.‘.’ Krlnndicck (:I,: Trouical sprut!: :I consitlcration of posslhle ctiologic mechan’isms with t!ml)h,rsis on ~~tero~lpol~gl~~tarnat~? metabolism. Trans Am (Olin (Zlimatol /Issoc 86: 11,1974. 110 1,uths:l I,. Salltini R. Brcwstcr C. c!t al.: Folatc hindinr IJV insohlhle ctrmponr~rlts of American and Puerto Rican [Gets. ,4k1bama I h,lctl Sci 2: 389. 1965. 111 Sczransnn \;I., !2’hcby h,lS. Bayless T!vI: hllqthnlogic effects nf fnlic ,ic:id and vitamin B,,>on the icilmal Icsion of tronical spruc’. Am J Path 49: I, l9;6. ’ ’ 114. Tomkins i\hl. James WP’I’,Drasiir US: f%lc:hrri.dcolonisation of ic:jun.tl l~ii~cosa in ,Icutr trnpical s1)r11,’Lancct 1: 5Y.

19%. 113 114

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Klipstoin f:A. Engert RF. Short HB: Entcrotoxigenicity of cnlonising cnliform bacteria in tropical spruc and blintlloop syn&ome. Lancet 8: 12, 1978.’ Hoffbrand AV. Stewart JS. Booth CC. et aI : Folate deficiency in Crohn’s disease: incitlencr~. i’athngencsis and treatment. Br h4ed J 2: 71. 19fi8.

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115. 116. 117. 118. 119. 120. 121. 122. 123.

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IN MALABSORPTION SYNDROME-LINDENBAUM

Franklin JL, Rosenberg IH: Impaired folic acid absorption in inflammatory bowel disease: effects of salicylazosulfapyridine (azulfidine). Gastroenterology 64: X7,1973. Cox EV. Mevnell Ml. Cooke WT: The folic acid excretion test in the sieatorrhea syndrome. Gastroenterology 35: 396, 1958. Booth CC: The metabolic effects of intestinal resection in man. Postarad Med 1 37: 725. 1961. Klipstein FL: Folate deficiendy secondary to disease of the intestinal tract. Bull NY Acad Med 42: 638,1966. Baker H, Thomson AD, Feingold S, et al.: Role of the jejunum in the absorption of folic acid and its polyglutamates. Am J Clin Nutr 22: 124.1969. Pavesio D, Bosio U: 11test di assorbimento dell’acido folico in alcune malattie intestinali infantili. Minerva Pediatrica 17: 1488,1965. Elsborg L, Bastrup-Madsen P: Folic acid absorption in various gastrointestinal disorders. Stand 1 Gastroent 11: 33,1976.Hoffbrand AV: Folate absorption. J Clin Path 24 (suppl5): 66, 1971. Hyde RD. Loehry CAEH: Folic acid malabsorption in cardiac failure. Gut 9: 717,1968.

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Elsborg L: Malabsorption of folic acid following partial gastrectomy. Stand ] Gastroent 9: 271.1974. Pitney WR, Joske RA, MacKinnon WL: Folic acid and other absorption tests in lymphosarcoma, chronic lymphocytic leukaemia, and some related conditions. J Clin Path 13: 440, 1960. Barrett CR Jr, Holt PR: Postgastrectomy blind loop syndrome. Am J Med 41: 6291966. Hermos JA, Adams WH, Liu YK, et al.: Mucosa of the small intestine in folate-deficient alcoholics. Ann Intern Med 76: 957,1972. Cook GC: Absorption of xylose, glucose, glycine, and folic (pteroylglutamic) acid in Zambian Africans with anaemia. Gut 7% Fio4,1976. Lindenbaum J: Metabolic effects of alcohol on the blood and bone marrow. Metabolic Aspects of Alcoholism, (Lieber CS, ed), Lancaster, MTP Press, 1977. p 215. Halsted CH, Robles EA. Mezey E: Intestinal malabsorption in folate-deficient alcoholics. Gastroenterology 64: 526, 1973.

131. Mekhjian SH, May ES: Acute and chronic effects of ethanol on fluid transport in the human small intestine. Gastroenterology 72: 1280, 1977.

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