0021-972X/82/5402-0381$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1982 by The Endocrine Society

Vol. 54, No. 2

Printed in U.S.A.

Vitamin B-12 (Cobalamin) Deficiency: A Heretofore Undescribed Control Mechanism for Plasma Corticosteroid-Binding Globulin Concentration in Man* RICHARD P. DOE, F. LOHRENZ,f G. RIVER, A. DOSHERHOLMEN, AND R. ROBERTS WITH THE TECHNICAL ASSISTANCE OF RITA LAMUSGA, MARY D. LEWIS, AND MARY K. TWITE The Metabolic-Endocrine and Hematology Sections, Minneapolis Veterans Administration Medical Center, the Endocrine Division, University of Minnesota Medical School, Minneapolis, Minnesota, 55417; and the Marshfield Clinic, Marshfield, Wisconsin 54449

a course parallel to that of CBG. The CBG concentration in two untreated patients had a normal increase in response to estrogen administration, similar to that described in CBG deficiency from other causes. No clue was found regarding the mechanism by which B12 influences the putative hepatic control of CBG concentration. It is speculated that at least two control points may be necessary for a normal CBG concentration. Patients with a genetic deficiency of CBG may also have to have cobalamin deficiency in order for them to reach a concentration approaching zero. (J Clin Endocrinol Metab 54:381, 1982)

ABSTRACT. Six of eight patients with Addisonian pernicious anemia were found to have diminished corticosteroid-binding globulin (CBG) concentrations, which returned within 2 weeks to normal after the im administration of cobalamin. T4-binding globulin was found to be completely normal before and after cobalamin replacement. Other estrogen-responsive proteins, i.e. ceruloplasmin, ai-trypsin inhibitor, haptoglobin, transferrin, and a2-macroglobulin, also did not follow CBG concentration in a parallel fashion. The immunoglobulins similarly did not follow

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these conditions, there is a modest but significant correlation with the decrease in albumin concentrations (r = 0.51; P < 0.05). Essentially all patients with a decreased CBG concentration on the basis of this type of disturbance have had a decrease in albumin concentration, but the reverse relationship is not consistent, i.e. a low albumin concentration does not necessarily mean that the CBG concentration is low (3). An inherited decrease (16) and a putative inherited increase in siblings (17) have also been reported. The absence of CBG from serum has not been reported to date in spite of a search by many laboratories and an exhaustive search by Rosner et al. (18) in 10,000 random individuals. The present study describes a decrease in CBG concentration which occurs in some, but not all, patients during cobalamin deficiency. One case of CBG elevation in uncomplicated dietary folic acid deficiency (normal B12) is also described. We believe that this study reports a heretofore unsuspected control mechanism for serum CBG concentration.

ERUM corticosteroid-binding globulin (CBG) concentrations in man have been well studied and found to be resistant to change in most clinical situations (1-4). The protein has been isolated and characterized previously (5-8). Extensive reviews of the clinical and biochemical status of the protein were published recently by Rosner (9) and Brien (10). Men and women normally have equivalent concentrations in the serum and both experience a 2- to 3-fold elevation after large dose estrogen administration or during pregnancy in females (1, 11-14). Although the baseline concentrations of CBG are equal in males and females, the threshold dosage required for elevation of the serum concentration is lower, and the plateau concentration of the dose-response curve for estrogen is higher in females (15). Decreases in the serum concentration of CBG have been common in disease states associated with a generalized disturbance in protein metabolism {i.e. nephrosis, cirrhosis, exudative enteropathy, amyloidosis, multiple myeloma, etc.) (3). In Received May 1, 1981. Address requests for reprints to: Richard P. Doe, M.D., Ph.D., Professor of Medicine and Endocrine Staff, Minneapolis Veterans Administration Medical Center, Minneapolis, Minnesota 55417. * This work was supported in part by grants from the V.A. and the Minnesota Chapter of the American Diabetes Association. f Deceased.

Materials and Methods The diagnosis of pernicious anemia was made by an abnormal Schilling test, bone marrow demonstration of typical megaloblastic changes, mean corpuscular volume of over 99 /im3, an 381

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anemia state of 5.3-12.3 g/dl, and reticulocyte and hemoglobin responses to vitamin Bi2 injections. Measurements of CBG (3), T4-binding globulin (TBG) (19), total protein (19), ceruloplasmin (19), a-trypsin inhibitor (19), transferrin (19), and haptoglobin (19) were made by previously described methodology. The interassay coefficient of variation for CBG was 3% (20). The CBG determinations were made in duplicate. Macroglobulin (a2), immunoglobulin G (IgG), IgA, and IgM were measured by radial immunodiffusion (Hyland Immunoplates, Coasta Mesa, CA). The experimentally determined normal range (95% confidence limits) for each protein is given at the bottom of Tables 1-3. The patient reported with pure dietary folic acid deficiency did not consume alcohol, had a normal fluid intake, normal hepatic function tests, no orthostatic change in blood pressure, normal skin turgor, moist mucous membranes, and a CBG elevation beyond that ordinarily seen with dehydration.

Results CBG concentration was diminished in six of eight patients studied during Bi2 deficiency (Table 1). Care was taken to study the baseline CBG concentration before 48 h had elapsed after the flushing dose of B12 (administered im) for the Schilling test. Repeat measurement at 3 months after the im administration of 1 mg cobalamin monthly showed that the CBG concentration had increased to the normal range in the patients with diminished concentration. The only two patients so studied had returned to a normal CBG serum concentration by 2 weeks after cobalamin administration, a period when multilobed polymorphonuclear leukocytes PMNs may still be present (21) and lymphocyte function may still be subnormal (22). The minimal time required for restoration of normal plasma CBG concentration was not ascertained. Two of three female patients with B12 deficiency had normal CBG concentrations. No change in CBG concentration occurred in response to cobalamin administration (1000 U monthly) in the two patients with B12 deficiency and normal CBG baseline values. The addition of cobalamin (1 mg) to serum (in vitro) with a low CBG concentration had no effect on the CBG measurement. Five male patients had low CBG serum concentrations, and all returned to normal after cobalamin replacement. Two patients (patients 4 and 5) with diminished CBG concentrations and untreated pernicious anemia responded normally to 100 /xg ethinyl estradiol daily for 1 week (CBG rose from a mean of 24 to 55 mg/liter postestrogen) without cobalamin replacement. A ninth patient with severe dietary serum folic acid deficiency but a normal serum cobalamin concentration had an elevated CBG concentration, which returned to normal after folic acid replacement. Simultaneous study of another estrogen-responsive serum protein in the CBG-deficient patients (Table 1),

J C E & M • 1982 Vol 54 • No 2

showed serum TBG to be normal before and after B12 replacement. Three of four CBG-deficient patients so examined had diminished ceruloplasmin concentrations, which rose to normal after B12 injections (Table 2). A normal ceruloplasmin response to estrogen was also noted before B12 administration in the two patients studied above for CBG response. ai-Trypsin inhibitor was low in three of four patients studied; one of these failed to return to normal after B12, treatment, and haptoglobin was low in two of four patients. On the other hand, 0:2macroglobulin and ceruloplasmin were elevated in one of four patients, and transferrin serum concentrations were normal, (elevated in one patient post-Bi2). Serum Ig had a mixed response without a discernible pattern (Table 3). IgG was elevated in four of six patients, IgA was elevated in four of six patients (decreased in one), and IgM was elevated in two patients and decreased in three. Patients with abnormal Ig concentrations (including a2-macroglobulin) also failed to consistently have a change to normal in these proteins after B12 replacement in the period of time studied. The propositus in the previously described kindred with a genetic decrease in CBG (16) was restudied and found to have normal cobalamin absorption and concentration in the blood. Discussion Clinical clues concerning the biological role of CBG revolve almost entirely around the changes which consistently occur in plasma before puberty (1, 15, 23) or during pregnancy in the maternal and fetal compartment (1-3), its presence in human milk (24), and the possibility that it may influence target tissue availability of cortisol separate from the receptor effect (25-27). The increase during pregnancy occurs almost exclusively in mammals with hemochorial placentation (maternal blood is in direct contact with the fetal trophoblast). Mammals with endothelial-chorial placentation (a maternal membrane is interposed between the maternal circulation and the fetal trophoblast) do not obtain an increase in CBG during pregnancy (4). The additional membrane barrier in the latter type of placentation might easily have immunological implications relative to the foreign body burden placed upon the mother by the fetus. The present report describes a heretofore undescribed control mechanism for CBG concentration, namely adequate blood and/or tissue levels of vitamin B12. The overall significance of this finding remains open to speculation. It may be that it represents merely one aspect of a generalized disturbance in the maintenance of serum protein concentrations during BJ2 deficiency. However, all previously described decreases in CBG concentration have been associated with diminished plasma albumin

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383

CBG IN VITAMIN B-12 DEFICIENCY

TABLE 1. Illustration of the low CBG concentration in some patients with B ]2 deficiency and the correction with 3 months of parenteral B ]2 administration Patient no. (sex) KM) Pre Post 2(M) Pre

Post 3(M) Pre

Post 4(M) Pre

Post 5(F) Pre

Post

CBG(mg/ liter)

TBG( M gT 4 bound/dl)

,

20 35

20 23

3.5

6.0

128

6900

Ketosis-prone diabetes, absent Achilles reflex

26 35

26 24

3.9

7.2

125

2800

None

23 31

21 23

3.7

8.3

119

7400

Ketosis-prone diabetes

24 38

26 22

4.6

8.3

130

1200

Absent Achilles reflex, absent vibration sense

25 33

22 21

3.0

5.3

100

3100

Ketosis-prone diabetes

32 35

19 21

4.5

9.3

107

4200

Previous treated Graves' disease

28 35

18 21

3.1

12.3

112

2900

None

32 31

25 21

3.2

5.9

123

2200

Renal insufficiency

52 43

22 20

2.9

4.9

140

4000

Malnutrition (1 yr)

3.5-5.3

12-17

80-93

"

r

WBC (mmJ)

Other

6(F) Pre

Post 7(M) Pre

Post 8(F) Pre

Post 9(M)° Pre

Post Normal range (mean ± 2SD)

28-42

13-S

The normal TBG concentration illustrates that this is not a generalized phenomenon related to estrogen-responsive (or steroid-responsive) serum proteins. Alb, Albumin; HB, hemoglobin; MCV, mean corpuscular volume; WBC, white blood cells. Pre, Before B12 treatment; Post, after B12 treatment (except for patient 9, where they refer to before and after feeding). " A patient with severe uncomplicated folic acid deficiency but normal B]2 serum concentration and absorption and elevated CBG concentration (see Materials and Methods).

concentrations (see Introduction) and so the phenomenon would be unique in that regard. In any event, a somewhat different medley of proteins is affected when compared to estrogen-induced changes in serum proteins (19), since a significant decrease in TBG was not detected. Two other estrogen-responsive serum proteins, i.e. ceruloplasmin and the ai-trypsin inhibitor, increased in three of four and in two of four patients, respectively, with B12 treatment. Interpretation of changes in these latter estrogen-responsive serum proteins is complicated by the fact that both are acute phase reactants. This confoundation is not encountered with CBG and TBG. A time curve for the recovery of plasma CBG concentration to normal during the first 2 weeks after cobalamin injection has not been established, and so it would seem to be important to look for the abnormality before the large flushing injection of cobalamin used in the Schilling test, i.e. before the diagnosis is confirmed. Patients with B12 deficiency may or may not present

with associated folic acid deficiency and/or diminished methionine blood levels. Pure folic acid deficiency is difficult to study in the patients in our hospital, since it is ordinarily associated with liver damage or other serious systemic disease known to influence CBG concentration. The one patient with elevated CBG due to dietary folic acid deficiency raises a question as to what would happen to the CBG concentration if severe combined cobalamin and folic acid deficiency were present concurrently. The influence of possible methionine deficiency cannot be effectively speculated upon by the authors. Vitamin B12 deficiency appears to cause megaloblastic anemia, granulopenia, and lymphopenia as the result of an effect on folate metabolism which indirectly reduces thymidylate synthesis from deoxyuridylic acid, a key step in DNA synthesis (28). The subtle difference in folate coenzyme concentrations in cobalamin deficiency vs. folic acid deficiency could conceivably lead to a difference in their effect upon protein-forming systems. Although de-

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TABLE 2. Changes in estrogen-responsive-acute phase reactive serum protein concentrations in four patients from Table 1 Ceruloplasmin (mg/dl)

Patient no.

ai-Tryp- a2-Macsin in- roglobuhibitor lin (mg/ (mg/dl) dl)

Haptoglobin (mg/dl)

Transferrin (mg/dl)

Pre B 12 Post B12

22 39

132 152

500 415

133 25

350 435

Pre B 12

Post B12

24 33

122 122

200 248

30 102

255 310

Pre B12 Post B12

59 32

191 175

230 310

86 177

225 270

Pre B12 Post B12

20 34

149 160

268 330

0 70

150 182

20-35

150-200

156-353

40-170

146-414

Normal / r a n g e (mean ± 2 SD)

TABLE 3. Baseline and postcobalamin replacement values for serum Igs in six patients from Table 1 Patient no.

IgG (mg/dl)

IgA (mg/dl)

IgM (mg/dl)

Pre B,2 Post B12

2450 2850

440 460

185 110

Pre B12 Post B12

2400 2400

72 105

71 92

Pre B 12 Post B12

1225 800

420 400

49 105

4 Pre Bi 2

1750

480

100

5 PreBi2

950

488

38

6 Pre B12

1750

380

172

700-1500

150-350

75-125

2

3

Normal range (mean ± SD)

creased synthesis of CBG would seem to be the most likely mechanism to achieve a decrease in CBG in pernicious anemia, an accelerated catabolism of transcortin remains a possibility. The liver stores a 3- to 5-yr supply of cobalamin for use by the bone marrow (and nervous system), but no effect of cobalamin upon hepatic metabolism has been reported. Although long suspected, it is only recently that conclusive evidence has been reported by Weiser et al. (29) that the liver is the site of CBG synthesis. In 1973, Werthamer et al. (30), using immunofluorescence, detected a CBG-like protein in the cytoplasm of human lymphocytes (30). This unconfirmed observation provides the only described cellular meeting place for CBG and Bi2 deficiency to date. Patients with cobalamin de-

J C E & M • 1982 Vol 54 • No 2

ficiency secondary to a genetic absence of the Bi2 carrier protein transcobalamin II frequently have deficient protein synthesis, i.e. virtually absent Igs with a return to normal after cobalamin replacement (31). However, here again, it is lymphocyte protein formation that is affected, i.e. in the mature B-lymphocyte or plasma cell. However, deficient hepatic synthesis would seem to be the most likely cause of the diminished serum CBG concentration during cobalamin deficiency. Previous studies by others have indicated that pernicious anemia was common (5%) in patients with common variable hypogammaglobulinemia (32), Bruton X-linked agammaglobulinemia (33), isolated IgA deficiency (34), and other putative autoimmune diseases such as Hashimoto's thyroiditis, Graves disease, ketosis prone diabetes mellitus, vitiligo, etc. (35). The present patients had both mild decreases and increases in Ig concentrations without a discernable pattern and without a definite return to normal after B12 replacement. Contrary to the data on TBG deficiency, no report of a patient with absent CBG in the serum exists in the literature. This may be because the defect is rare [and not discovered because the number of serum T4 determinations exceeds by an appropriate amount the number of critical cortisol determinations performed on patients (made especially difficult to detect because of cortisol's wide circadian range) and so the chance to discover a clinical disparity is greatly diminished], or the absence of CBG may be a lethal mutation. The present paper raises an alternate explanation, the possibility of multiple (at least two) control points required for the maintenance of a normal CBG concentration in the blood. The kindred with a genetic decrease in CBG concentrations of one half to one third the normal level (14) might have had to have cobalamin deficiency in order to reach zero CBG concentrations. Acknowledgments The aid of JoAnn Tallman and Charlotte Swanson in the preparation of the manuscript was greatly appreciated.

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CBG IN VITAMIN B-12 DEFICIENCY 6. Slaunwhite Jr WR, Schneider S, Wissler FC, Sandberg AA 1966 Transcortin: a corticosteroid-binding protein of plasma. IX. Isolation and characterization. Biochemistry 5:3527 7. Muldoon TG, Westphal U 1967 Steroid-protein interactions. XV. Isolation and characterization of corticosteroid-binding globulin from human plasma. J Biol Chem 242:5636 8. Rosner W, Bradlow HL 1971 Purification of corticosteroid-binding globulin from human and cavian plasma. J Clin Endocrinol Metab 33:193 9. Rosner W 1976 The binding of steroid hormones in human serum. Prog Clin Biol Res 5:377 10. Brien TG 1981 Human corticosteroid binding globulin. Clin Endocrinol (Oxf) 14:193 11. Gemzell CA 1953 Blood levels of 17-hydroxycorticosteroids in normal pregnancy. J Clin Endocrinol Metab 13:898 12. Wallace EZ, Carter AC 1960 Studies on the mechanism of the plasma 17-hydroxycorticosteroid elevation induced in man by estrogens. J Clin Invest 39:601 13. Mills IH, Schedl HP, Chen Jr PS, Bartter FC 1960 The effect of estrogen administration on the metabolism and protein binding of hydrocortisone. J Clin Endocrinol Metab 20:515 14. Doe RP, Zinneman HH, Flink EB, Ulstrom RA 1960 Significance of the concentration of nonprotein-bound cortisol in normal subjects, Cushing's syndrome, pregnancy and during estrogen therapy. J Clin Endocrinol Metab 20:1484 15. Musa BU, Seal US, Doe RP 1965 Serum protein alterations in women by synthetic estrogens. J Clin Endocrinol Metab 25:1163 16. Lohrenz FN, Seal US, Doe RP 1967 Adrenal function and serum protein concentrations in a kindred with decreased corticosteroidbinding globulin concentration. J Clin Endocrinol Metab 27:966 17. Lohrenz FN, Seal US, Doe RP 1968 Idiopathic or genetic elevation of corticosteroid-binding globulin? J Clin Endocrinol Metab 28:1073 18. Rosner W, Darmstadt RA, Toppel S 1974 A simple precipitin test for corticosteroid-binding globulin: use in a search for the absence of corticosteroid-binding globulin in 10,000 subjects. J Clin Endocrinol Metab 3:983 19. Doe RP, Mellinger GT, Swaim WR, Seal US 1967 Estrogen dosage effects on serum proteins: a longitudinal study. J Clin Endocrinol Metab 27:1981 20. Goldstein A 1967 Biostatistics. Macmillan, New York, p 36 21. Nath BJ, Lindenbaum J 1979 Persistence of neutrophil hyperseg-

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mentation during recovery from megaloblastic granuloporesis. Ann Intern Med 90:757 Das KC, Herbert V 1978 The lymphocyte as a marker of past nutritional status: presistence of abnormal lymphocyte deoxyuridine (dU) suppression test and chromosomes in patients with past deficiency of folate and vitamin B12. Br J Haematol 38:219 DeMoor P, Steeno O, Brosens I, Hendricks A 1966 Data on transcortin activity in human plasma as studied by gel filtration. J Clin Endocrinol Metab 26:71 Rosner W, Beers TC, Awas T, Khan MS 1976 Identification of corticosteroid binding globulin in human milk: measurement with a filter disc assay. J Clin Endocrinol Metab 42:1064 Keller H, Richardson UI, Yates FE 1969 Protein binding and the biological activity of corticosteroids: in-vivo induction of hepatic and pancreatic alanine aminotransferases by corticosteroids in normal and estogen treated rats. Endocrinology 84:49 Rosner W, Hochberg R 1972 Corticosteroid-binding globulin in the rat: isolation and studies of its influence on cortisol action in-vivo. Endocrinology 91:626 Dancis J, Jansen V, Levitz M, Rosner W 1978 Effect of protein binding on transfer and metabolism of cortisol in perfused human placenta. J Clin Endocrinol Metab 46:863 Friedkin M, Roberts D 1956 Conversion of uracil deoxyriboside to thymidine of deoxyribosenucleic acid. J Biol Chem 222:653 Weiser JN, Do V, Feldman D 1979 Synthesis and secretion of corticosteroid binding globulin by rat liver. J Clin Invest 63:461 Werthamer S, Samuels AJ, Amand L 1973 Identification and partial purification of "transcortin"-like protein within human lymphocytes J Biol Chem 248:6398 Hitzig WH, Landolt R, Miller G, Bodmer P 1974 Hereditary transcobalamin II deficiency: clinical findings in a new family. J Pediatr 85:622 Twomey JJ, Jordan PH, Laughter AH, Meuwissen HJ, Good RA 1970 The gastric disorder in immunoglobulin-deficient patients. Ann Intern Med 72:489 Douglas SD, Goldberg LS, Fudenberg HH, Goldberg SB 1970 A gammaglobulinemia and co-existent pernicious anemia. Clin Exp Immunol 6:181 Amann AJ, Hong R 1971 Selective IgA deficiency: presentation of 30 cases and a review of the literature. Medicine 30:223 Volpe R 1977 The role of autoimmunity in hypoendocrine and hyperendocrine function. Ann Intern Med 87:86

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