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Thiamine-responsive megaloblastic anemia
494
April, 1969 T h e Journal o] P E D I A T R I C S
Thiamine-responsive megaloblastic anemia An 11-year-old Caucasian girl is presented who had a megaloblastic anemia responsive only to thiamine. Other abnormalities inekuded diabetes mellitus, aminoaciduria, and sensorineural dea[ness. Initially the anemia, re#actory to vitamin Ble and ]olic acid
therapy, responded to administration o] a multiple vitamin preparation. Vitamin supplementation was withdrawn ]ollowed by a recurrence o] anemia 3 ~ months later. The implicated vitamins were then administered sequentially. A retieuloeytosis [ollowed administration o] thiamine. Anemia again recurred 4 months a#er cessation o] vitamin supplementation. On this occasion the anemia was corrected by the oral administration o[ 20 rag. thiamine daily. Thiamine blood levels and activities o] 3 thiamine-dependent enzymes o] the patient's blood cells were normal, excluding a generalized de]eet o] thiamine metabolism. The patient there]ore appeared to have a thiamine-dependent megaloblastic anemia. This represents the first demonstration o] a role ]or this vitamin in D N A metabolism.
Lon E. Rogers, M.D., F. Stanley Porter, M.D.,* and James B. Sidbury, Jr., M.D. DURHAM~
N. G.
MEOALOBLASTIC A N E M IA S of childhood are rare and are most often the result of vitamin B12 or folic acid deficiency. R a r e r causes are hereditary orotic aciduria, 1 sideroblastic anemias, 2 and malignancies of the di G u g l i e l m o variety? This report presents a patient whose megaloblastic anemia was unresponsive to folic acid and vitamin B12 but responded to the administration of thiamine. From the Department o] Pediatrics, Duke University Medical Center. This work was supported by National Institutes o] Health Grants Nos. 5 TO1 H E 05478, 2 R O 1 A M 06815, and MO1 FR 30. Presented in part at the meeting o] the Society ]or Pediatric Research, Atlantic City, New Jersey, April 28, 1967. *Reprint requests: Department of Ped~atrlcs, Duke Un~verslty Medical Center, Durham, N. C. 27706 Vol. 74, No. 4, pp. 494-504
Anemia recurred twice during periods of normal dietary thiamine intake and was corrected by supplementation with the vitamin. This unique response of a megaloblastic anemia to thiamine led to additional studies of the patient, including the activities of known thiamine-dependent enzymes, blood thiamine levels, and the incorporation of nucleosides into lymphocyte nucleic acids.
CASE REPORT AND CLINICAL STUDIES The patient, an l l-year-old Caucasian girl, was born November 11, 1954, following an apparently normal gestation. The birth weight was 3.6 kilograms; growth and development during infancy were normal. The family history revealed no relatives with deafness, diabetes, or
Volume 74 Number 4
MegaIoblastic anemia
49 5
Fig. 2. Bone marrow showing megaloblasts.
Fig. 1. Peripheral blood smear showing macrocytosis, anisocytosis, and poikilocytosis. unexplained anemia. At 3 years of age the patient developed diabetes mellitus, and at 4 ~ years of age hearing loss was first noticed. Bilateral sensorineural deafness was complete by age 7I~ years. Hemoglobin determinations done at age 1, 7, and 10 7/12 years were 10.5, 11.5, and 10 Gm. per cent, respectively. In February, 1966, a severe anemia was first detected; the hemoglobin was 6.0 Gm. per cent. Oral iron was administered for 2 89 months without response, and a packed erythrocyte transfusion was then given. On April 5, 1966, a smear of a bone marrow aspirate revealed megaloblastic changes. A serum vitamin B12 level measured 756 /z/zg per milliliter (Bioscience Laboratories). Oral folic acid (5 rag. 3 times daily for 2 weeks) and parenteral vitamin B12 (100/Lg daily for 1 week) were administered without hematologic response. The patient was then referred for evaluation. Upon admission to Duke University Medical Center Hospital on June 28, 1968, the patient appeared pale and was obviously deaf. The weight was 32 kilograms, the height 134 cm. The tympanic membranes were normal in appearance. A soft precordial heroic murmur was audible; otherwise, the heart was normal. The liver, spleen, and lymph nodes were not palpably enlarged. There were no edema, peripheral neuropathies, or other signs of beriberi.
The results of the admitting laboratory work were as follows: hemoglobin 5.8 Gm. per cent, hematocrit 19 per cent, leukocytes 3,500 per cubic millimeter, platelets 145,000 per cubic millimeter, reticulocytes 4 per cent. The differential leukocyte count was: 4 bands, 49 polymorphonuclear forms, 32 lymphocytes, 11 monocytes, 4 eosinophils. The peripheral blood smear (Fig. 1) revealed moderate poikilocytosis and anisocytosis with numerous macrocytes, giant platelets, and a slight degree of nuclear hypersegmentation of the granulocytes. A smear of a bone marrow aspirate showed erythroid hyperplasia with numerous megaloblasts (Fig. 2). Little stainable iron and no ring sideroblasts were present. Hemoglobin electrophoresis revealed A hemoglobin. The sickle cell preparation was negative. Erythrocyte autohemolysis was 0.17 per cent at 24 hours, 0.84 per cent at 48 hours. Incubated erythrocyte osmotic fragility was normal. The serum iron concentration was 115 /~g per cent; the total iron binding capacity was 300/~g per cent. Beginning on July 3, 1966, Berocca-C, ~ 2 ml., was administered parenterally once daily for 5 consecutive days. A reticulocyte response of 10 per cent was first apparent on the fourth clay of treatment and reached a peak of 19 per cent on ~Berocca-C (Roche) contains per 2 ml.: thiamine HCI 10 mg., riboflavin I0 rag., pyrldoxine 20 rag., niacinamlde 80 rag., paathenol 20 mg., biotin 0.2 rag., aseorbie acid 100 mg.
49 6
Rogers, Porter, and Sidbury
the eighth day of treatment. The reticulocytosis
was followed by a rise in hemoglobin to a maximum of 9.8 Gin. per cent on July 19, 1966 (Fig. 3). At the same time the leukopenia and thrombocytopenia were corrected. A posttreatment bone marrow obtained on July 13, 1966, showed improvement but not complete resolution of the megaloblastic changes. Deca-Vi-Sol,t 1 chewable tablet daily, was administered from July 6 through July 17, 1966. On July 6, 1966 (after 3 days treatment with Berocca-C) the patient experienced transient hypoglycemia. The diabetes had been well controlled with an 1,800 calorie diet, NPH insulin 40 units, and regular insulin 90 units per day. During the following 10 days, however, hypoglycemic attacks necessitated a decrease of insulin dosage to approximately one half the pretreatment level. Supplemental vitamins were not administered after discharge from the hospital, and a balanced 1,800 calorie diabetic diet was continued unchanged. Follow-up laboratory tests on August 2, 1966, and August 29, 1966, revealed no change in the hematologic status. Blood smears continued to show moderate anisocytosis and poikilocytosis. By late August the insulin requirements had increased to 50 units N.P.H. insulin daily. Signs of hematologic relapse were present on Oct. 24, 1966, approximately 3~2 months after the first remission. The hemoglobin was 7.8 Gin. per cent, hematocrit 24 per cent, leukocyte count 5,400 per cubic millimeter, reticulocytes 1.8 per cent, platelets 107,500 per cubic millimeter, erythrocytes 2.34 • 106 per cubic millimeter, MCV 102 cubic microns, M C H 33 /~/xg, MCHC 33 per cent. The morphologic abnormalities of the peripheral blood smear were again present. The bone marrow was frankly megaloblastic. Stainable iron was present, but there were no ring sideroblasts. During the second admission to the hospital the vitamins present in Berocca-C were given in a sequential manner to ascertain which vitamin, if any, had" produced the original response. The vitamins given, the reticulocytosis, and the hemoglobin'response are shown in Fig. 4. A reticulocytosis followed the administration of thiamine. The reticulocyte count peaked at 22 per cent, 9 days after the initiation of thiamine, and was followed by a rise of hemoglobin to 13.6 Gin. "~Deca-Vi-Sol (Mead Johnson) contains per tablet: vitam i n A 4,000 units, vitamin D 400 units, ascorbic acid 75 rag., thiamine 1.2 rag., riboflavin 1.5 m g . , niacinamide 15 m g . , pyrldoxlne 1.2 m g . , pantothenlc acid 5 m g . , v i t a m i n B~s 3 /tg,
biotin 40 #g, ferrous fumarate 40 mg.
The Journal of Pediatrics April 1969
per cent. The borderline thrombocytopenia and leukopenia were also corrected, but insuliri requirements did not decrease as Previously. In an attempt to again induce improvement of the diabetes, biotin, pantothenic acid, and finally Berocca-C were administered. The insulin requirements did not change. Following the last injection of Berocca-C on Jan. 5, 1967, all supplemental vitamins were discontinued. No medication except insulin was taken. Hematologic remission continued for 4 months until May, 1967. Upon readmission in relapse, the hemoglobin was 8.5 Gin. per cent, erythrocytes 2.46 • 106 per cubic millimeter, leukocytes 6,000 per cubic millimeter, hematocrit 26 per cent, reticulocytes 6.3 per cent, platelets 180,000 per cubic millimeter, MCV 106 cubic microns, M C H 35/z/zg, MCHC 33 per cent. The bone marrow and peripheral blood smears showed the previously described abnormalities. Administration of oral uridine 1.0 Gin. per day for 5 days (May 11 to 15, 1967) and yeast DNA 1.5 Gm. per day for 5 days (May 16 to 20, 1967) failed to produce a hematologic response. Following these trials, thiamine, 20 rag. per day orally, was begun on May 22, 1967. Reticulocytosis began on the fourth day of treatment and peaked at 14.5 per cent on the eleventh day. The hemoglobin rose to 13.7 Gin. per cent by the twenty-eighth day of thiamine administration (Fig. 5). The leukocyte and platelet counts
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Volume 74 Number 4
Megaloblastic anemia
were within the normal range during the second relapse and did not change with treatment. Following each of the hematologic responses, the abnormalities of the peripheral blood smear were not completely corrected; anisocytosis and poikilocytosis of erythrocytes persisted. In addition to the anemia, diabetes, and deafness, other abnormalities were present. An electroencephalogram obtained during hematologic relapse showed the following features: spike wave abnormalities of the temporal region of a 55~2 per second frequency and not exceeding 100 microvolts amplitude. In the parietal region, 6 per second high voltage discharges with amplitudes in the 200 microvolt range were seen. There was no alteration of this bizarre tracing with therapy. The patient had never had any seizure activity. Although her motor development was normal, her intellectual development was somewhat impaired. The deafness and unwillingness
to cooperate with simple instructions had made an exact evaluation of intelligence impossible, but her achievement at school was on a kindergarten level. Other laboratory findings included the following: serum sodium 136 mEq. per liter, potassium 5 mEq. per liter, carbon dioxide 28 mEq. per liter, calcium 9.8 mg. per cent, phosphorus 4.1 mg. per cent; blood urea nitrogen 9 mg. per cent; total serum protein 7.8 Gin. per cent, albumin 4.6 Gm. per cent, globulin 3.2 Gm. per cent, IgG 1,150 mg. per cent, IgA 100 mg. per cent, and IgM 59 mg. per cent, PBI 6.9 /xg per cent, serum glutamic-oxalacetic transaminase 15.4 units, serum glutamic-pyruvic transaminase 40 units, zinc turbidity 16 units, alkaline phosphatase 7.0 Bodanski units, and prothrombin time 83 per cent of normal. Routine urinalysis revealed: specific gravity 1.005, pH 6.5; the reactions for protein, sugar, and acetone were
I~- Thiomine----I Biotin Berocco-C - 24 Niocin I I I I I I Pontothenic Pantothenic 22 ir Acid Acid I I I I H
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Fig. 4. Reticulocytosis and rise of hemoglobin following administration of thiamine. Lack of response to pyridoxine, riboflavin, vitamin C, niacin, and pantothenic acid is shown. The daily dosages and dates of administration of the vitamins are as follows: pyridoxine 25 rag., Nov. 8-13, 1966; riboflavin 30 rag., Nov. 14-22; ascorbic acid 50 rag., Nov. 19-23; pantothenic acid 950 rag., Nov. 24-Dec. 2; niacin 50 rag., Nov. 30-Dec. 4; thiamlne 50 nag., Dec. 5-16; thiamine 95 mg., Dec. 17-22; biotin 10 rag., Dec. 19-16; pantothenic acid 63 rag., Dec. 20-22; Berocca-C 2 ml., Dec. 28-31; Berocca-C 1 ml., Jan. 1-5, 1967. All vitamins were given intramuscularly except biotin and riboflavin, which were taken orally.
4 98
Rogers, Porter, and Sidbury
The Journal of Pediatrics April 1969
,
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Fig. 5. Reticulocytosis and rise of hemoglobin following administration of oral thiamine 20 mg. per day. Lack of response to uridine and DNA is shown.
negative; the microscopic examination revealed an occasional epithelial cell. Urinary FeCla, DNPH, and CTAB tests were negative. Additional studies of special interest included the following: Activities of the erythrocyte enzymes, orotidylic pyrophosphorylase, and orotidylic decarboxylase4 were determined and were not decreased. Urinary screening test for hereditary orotic aciduria 5 was also negative. Urinary formiminoglutamic acid excretion was not excessive after histidine load. 6 Urinary excretion of methylmalonic acid and xanthurenic acid during relapse was also normal. The diabetes was unresponsive to administration of tolbutamide. An electrocardio.gram during relapse was normal. Following the last remission the patient took 20 rag. thiamine orally per day and maintained a normal hemoglobin for more than one year. SPECIAL STUDIES The response of the megaloblastic anemia to thiamine suggested at least two possibilities regarding the underlying defect in this patient. The disorder might involve primarily the metabolism of thiamine and result
secondarily in the malfunction of numerous thiamine-dependent enzymatic processes , or the defect may lie in a specific thiaminedependent enzyme so that it alone operates suboptimally. To investigate these possibilities, the activities of 3 thiamine-dependent enzymes were assayed and blood thiamine levels were measured. T h e megaloblastic features of the anemia led to studies of the incorporation of uridine and thymidine by lymphocytes and of lymphocyte transformation following stimulation by phytohemagglutinin. The finding of elevated urinary amino acids led to their quantitation in urine and plasma. METHODS
Erythrocyte transketolase. The method used was that reported by Wolfe and associates. ~ Erythrocyte transketolase is assayed by cycling 2-C*4-glucose through the pentose shunt of the glycolytic pathway, a process requiring the activity of this thiamine-de-
Volume 74 Number 4
pendent enzyme. Loss of enzymatic activity is reflected by a deficient production of carbon dioxide-C ~* during the second transit of the sugar molecule through the pentose pathway. An erythrocyte suspension of known concentration was prepared from heparinized blood by centrifugation and by removal of plasma and buffy coat, followed by resuspension in an equal volume of buffer of p H 7.4 constituted as follows: 20 raM. sodium phosphate, 5 mM. magnesium chloride, 15 raM. sodium chloride, and 4 raM. potassium chloride. This buffer was also used in the other enzyme assays to be described. The reaction mixture consisted of a 0.4 ml. volume of the erythrocyte suspension and 0.2 ml. of aqueous 0.025 per cent methylene blue with 0.06/~c (5.06/~moles) 2-C 14glucose made up to a 1 ml. volume with buffer. Duplicate samples were run with and without thiamine hydrochloride in a final concentration of 10-s M. A blank was prepared by omitting the erythrocyte suspension. The samples were incubated at 37 ~ C. in a shaking water bath for 3 hours. The C ~4 02 was collected in hydroxide of Hyamine which was counted in a toluene-phosphor mixture in a Packard Tri-Carb Liquid Scintillation Counter. The results are expressed as mp.moles of COs per l0 s erythrocytes per hour. This and the following assays described were performed with blood taken from the patient during relapse. No supplemental thiamine was b e i n g administered. The enzymatic activities of control blood samples were determined simultaneously. Leukocyte pyruvlc dehydrogenase. Pyruvate is decarboxylated by leukocyte pyruvie dehydrogenase to form acetyl-CoA and carbon dioxide. The activity of this thiaminedependent enzyme was assayed by the recovery of carbon dioxide-C 14 following the incubation of leukocytes with 1-C~4-pyruvate. A modification of the method published by Dreyfus and HauseP was used. Cell suspensions were prepared by removal of plasma from heparinized whole blood following centrifugation. Since erythrocytes lack this enzyme, no attempt was made to remove them.
Megaloblastic anemia
499
Sedimented cells were resuspended in a volume of the buffer described above to provide a concentration of leukocytes from 5,000 to 10,000 per cubic millimeter (Coulter Counter). A volume of 0.7 ml. of this suspension was combined with 0.4 ml. of the buffer containing 0.017 ~c (72 /xmoles) 1-C14-sodium pyruvate. From this point the assay was performed with and without thiamine, as described for the erythrocYte transketolase assay. Results are expressed as m~moles CO2 per 105 leukocytes per hour. Leukocyte a-ketoglutaric dehydrogenase. The carboxyl carbon of a-ketoglutaric acid is released as CO2 upon the conversion of this acid to succinyl-CoA in the presence of oe-ketoglutaric dehydrogenase. The activity of this thiamine-dependent enzyme was assayed by the collection of carbon dioxide-C 14 when labeled o~-ketoglutaric acid was used as the substrate. Leukocytes were prepared and counted as previously described. A 0.5 ml. volume of the suspension was added to 0.5 ml. of buffer containing 0.08 ~c (6.35 /~moles) 1,2-C-~4-o~-ketoglutaric acid. From this point the assay was performed with and without thiamine, as described for the transketolase assay. Enzymatic activity of a normal control sample was determined simultaneously. The results are expressed as m/~moles CO2 per 105 leukocytes per hour. Blood thiamine assay: Blood thiamine was assayed according to the method of HaugenP Thiamine pyrophosphate was hydrolyzed with phosphatase to form the free vitamin which was oxidized by potassium ferricyanide to the fluorescent thiochrome. The fluorescence was measured after extraction of the thiochrome into isobutanol. Powdered Takadiastase (Parke-Davis) was used as a source of phosphatase? ~ All samples were measured in duplicate. Leukocyte transformation and incorporation studies. The basic medium was Eagle's M E M ~x with Earle's balanced salt solution. The p i t was adjusted to 7.4 with 5 per cent sodium bicarbonate. For the studies designed to demonstrate thiamine dependency, the medium was identical except that thiamine
500
Rogers, Porter, and Sidbury
was omitted in preparation. Phytohemagglutinin P was obtained from Difco Laboratories. Plasmagel was obtained from the Laboratorie Roger Bellon. Ha-thymidine and HS-uridine were obtained from New England Nuclear Corp. I-Ieparinized blood was obtained from the patient when in relapse and mixed with volume of Plasmagel for sedimentation of the red ceils. One milliliter of cells suspended in the plasma-Plasrnagel mixture was transferred to a stoppered test tube containing 4 ml. of culture medium containing peniciIlin and streptomycin as the antibiotics. Phytohemagglutinin P was added in a volume of 0.1 ml. Cultures were incubated at 37 ~ C. for 72 hours. Velban (Grand Island Biological Co.) was added two hours before harvesting to effect metaphase arrest. The cells were harvested and prepared after the method of Moorhead and associates. 12 In the studies with H3-thymidine and H 3uridine incorporation, 4 ~c of the tritiated nucleotide was added to the media 6 hours before harvesting. After the slides were fixed and stained, they were dipped in Kodak nuclear tract emulsion (NBT2) and kept in the Cambridge box for 6 weeks before being developed. Aminoacid studies. Aminoacid analyses were performed on aliquot parts of 24 hour urine collections. Plasma aminoacids were determined after precipitation of the protein with picric acid which was removed by passage through a column of Dowex 2 x 10. The aminoacids were separated and quantitated with a Beckman Spinco amino acid analyzer using the conditions described by Spackman and associates33 Loading icests were performed by oral administration of 150 mg. per kilogram of the amino acids lysine and valine; the tests were carried out before and during therapy with thiamine. Because of technical limitations only a 3 hour sample was obtained after loading, this being a time when the values would be expected to have returned to near control levels in the normal individual, but could be expected to be elevated if there were delayed clearance. 14
The Journal of Pediatrics April 1969
RESULTS Erythrocyte transketolase. Averaged values of duplicate determinations are expressed as m,amoles CO2 per l0 s erythrocytes per hour. Activities for the patient's erythrocyte transketolase were 12.0 with thiamine added to the reaction mix, and 11.9 without added thiamine. Corresponding activities for the control subject were 6.3 and 4.9, respectively. Leukocyte pyruvic dehydrogenase. Results are expressed as m~moles CO2 per 105 leukocytes per hour. Averaged values for activity of the pyruvic dehydrogenase for the patient were 1.6 with and 1.5 without added thiamine. For the control the respective values were 2.0 and 2.3. Leukocyte ~-ketoglutaric dehydrogenase. With enzyme activities expressed as m~moles CO2 per 10 5 leukocytes per hour, averaged values for duplicate samples for the patient were 0.45 with added thiamine and 0.29 without added thiamine. For the control the values were 0.51 with added thiamine and 0.38 without added thiamine. Blood thiamine assay. Values for duplicate samples of total blood thiamine for the patient during relapse averaged 4.8/~g per cent. Normal values by this method range from 2.48 to 10.77 ~g per cent with an average of 5.28 /~g per cent."
Leukocyte transformation and incorporation studies. The studies of lymphocyte transformation consequent to phytohemagglutinin stimulation were indistinguishable in the media with and without added thiamine in terms of the percentage of large lymphocytes and mitotic figures. No difference was demonstrated in tritiated thymidine or uridine incorporation with or without added thiamine in the media, as judged from the number of grains per cell. Normal control cells run simultaneously could not be distinguished from the patient's ceils by the response given. Amino acid studies. The urinary content of amino acids in a 24 hour specimen were: valine 61 mg. per 24 hours before therapy and 47 rag. per 24 hours during therapy with thiamine, compared with a normal range of 4 to 6 rag. per 24 hours; lysine 74 mg. per 24 hours before and 203 mg. per 24 hours
Volume 74 Number 4
Megaloblastie anemia
50 1
Table I. Plasma amino acids (milligrams per 100 ml.). The quantitative results of plasma aminoacids in the control period and 3 hours after valine and lysine loading (150 rag. per kilogram) are presented. The loading tests were run before therapy was instituted and several days after high doses of thiamine had been administered. Average adult control values are listed for comparison ~2
Amino acid Aspartic acid Threonine Serine Proline Glutamic acid Glycine Alanine Half eystine Methionine Isoleueine Leucine Tyroslne Phenylalanine Histidine Arginine Lyslne Valine
Fasting Trace 0.76 0.78 0.99 0.81 1.20 1.35 Trace Trace Trace 0.81 Trace Trace 1.35 Trace 1.26 1.46
Valine load Lysine load Be[ore thiAfter thiBe[ore thiAlter thiamine therapy amine therapy amine therapy amine therapy 0.40 Trace 0.09 Trace 3.35 1.49 1.99 2.29 3.41 L72 1.89 1.69 2.7O 0 4.62 0 4.16 1.04 2.43 1.10 3.48 1.67 4.24 1.40 7.97 4.29 8.44 4.9 0 Trace Trace Trace 1.06 0.55 Trace 0.37 2.98 1.76 0.22 1.46 7.37 1.98 1.69 1.99 0 1.2 0 1.07 0 0.83 0 0.69 3.52 1.30 1.01 1.65 2.56 Trace Trace 0.91 20 4.36 3.98 1.68 38.16 27.81 9.75 3.2
during therapy, compared with normal range of 7 to 48 mg. per 24 hours. 15 The other amino acids were present in normal quantities. The finding of elevated valine and lysine levels in the urine prompted the investigation of the plasma levels and loading response to these 2 amino acids. The plasma sample was obtained before and 3 hours after the load to discriminate abnormal clearance of the loaded dose from the normal. 1~ The values obtained with the valine loading were abnormal and were the same before and after thiamine therapy. Lysine clearance was definitely abnormal before, but normal after, therapy was instituted. There was a small but significant rise in proline with both loading tests before therapy, and a decrease to undetectable levels in response to these loading tests after therapy was commenced (Table I ) . DISCUSSION
All of the usual causes of megaloblastic anemia may be excluded in this patient. .Classical pernicious anemia is excluded by
Average adult control ee
0.5 2.0 1.4 2.6 1.1 2.8 3.8 1.0 0.35 1.6 2.0 1.4 1.6 1.7 2.1 2.8 3.0
the normal serum vitamin BI~ level, the failure of response to parenteral vitamin B~, and the normal urinary excretion of methylmalonie acid. Folic acid deficiency is similarly excluded by the lack of response to folate therapy and by the normal urinary excretion of formiminoglutamic acid. di Guglielmo's erythremic myelosis is a progressive, malignant process unresponsive to known hematinics and usually terminates fatally within months? Hereditary orotic aeiduria is excluded by assays of the activities of the enzymes, orotidylic pyrophosphorylase and orotidylic decarboxylase, and by the urinary screening test for this disorder. 4' ~ Megaloblastic changes are aIso found in the bone marrow of some patients with sideroblastic anemia. Signs of folate deficiency and a partial response to this vitamin or to pyridoxine are sometimes observed. Characteristic findings are increased numbers of ringed sideroblasts in the marrow and an excessive excretion of urinary xanthurenie acid. The patient's anemia, on the other hand, was unresponsive to both pyridoxine and folic acid,
5 0 2 Rogers, Porter, and Sidbury
the bone marrow was not iron loaded, and the urinary excretion of xanthurenic acid and formiminoglutamic acid was not elevated. Isolated cases of megaloblastic anemia totally unresponsive to therapy or respondi n g to unusual therapeutic regimens have been reported. 16-19 These disorders, however, do not present the same features as the patient reported here. The response of this patient's anemia to the administration of thiamine is unique and suggests one of several possible derangements of thiamine metabolism: deficient intake, defective absorption, excessive excretion, inadequate utilization, or an increased requirement. The first three possibilities may be excluded for the following reasons: (1) Clinical signs of beriberi were not present, (2) blood thiamine levels were normal, (3) erythrocyte transketolase activity was not reduced. The transketolase assay is an especially sensitive indicator of thiamine deficiency, and low blood levels of this vitamin for any reason (decreased intake, inadequate absorption, or excessive excretion) would be expected to result in decreased activity, s~ Furthermore, anemia, if present at all, is a late manifestation of thiamine deprivation. The presence of a severe anemia without any other signs of beriberi is not readily explained on the basis of a generalized thiamine deficiency. Thiamine pyrophosphate is a cofactor for several essential enzymatic reactions such as the transketolation reaction and the decarboxylation of o~-ketoacids. In an attempt to exclude a generalized failure of thiamine utilization in this patient, 3 thiamine-dependent enzymes were assayed: erythrocyte transketolase, leukocyte pyruvic dehydrogenase, and leukocyte a-ketoglutaric dehydrogenase. The activities of these particular enzymes were found to be normal, and their activities were not significantly altered by the addition of thiamine hydrochloride in vitro. The finding of 3 normally functioning thiamine-dependent enzymes in this patient's blood cells demonstrates that the defect does not affect all enzymes utilizing this vitamin co factor.
The ]ournal o[ Pediatrics April 1969
The most reasonable explanation for the response of this child's anemia to thiamine is an increased requirement for this vitamin, a thiamine-dependent state. Whereas the ingestion of normal dietary amounts of thiamine was insufficient to prevent the development of anemia, it was corrected by the administration of amounts significantly greater than the accepted minimum daily requirement. This finding suggests that normal hematopoiesis is made possible in this child by the elevation of tissue thiamine concentrations. High thiamine intake may load tissues and expand stores 2 to 3 times normal, el but once supplementation is withdrawn, a prompt decrease of stores to pretreatment level is to be expected. In the studies of Brin, 2~ for example, human volunteers were given 5 mg. thiamine daily for 5 days, an amount adequate to replete all stores, and thereafter thiamine-poor diets were ingested. Biochemical evidence of thiamine deficiency was apparent 8 days later, and symptoms of depletion appeared within 21 days. Apparently so long as this patient's tissue level of thiamine could be maintained by supplementation at superphysiologic levels, normal hematopoiesis was possible. With physiologic levels of tissue thiamine, however, hematopoiesis was retarded and within weeks resulted in anemia. The response of the disorder to large but not to physiologic amounts of the vitamin has a parallel in pyridoxineresponsive anemia. 2 The megaloblastic features of the anemia suggest that the postulated defect involves the synthesis of nuclear material (DNA). Until now there has been no evidence to implicate thiamine in DNA synthesis. To study this possibility further, DNA and uridine were administered to the patient. In addition, lymphocytes obtained during hematologic relapse were incubated with labeled thymidine and uridine and examined autoradiographically. The administration of DNA and uridine failed to produce a hematologic response, and the patient's lymphocytes incorporated thymidine and uridine normally. In addition, Iymphocytes s t i m u 1 a t e d with
Volume 74 Number 4
phytohemagglutinin underwent characteristic blast transformation. From these studies the mechanism of impairment of nucleic acid metabolism accounting for the megaloblastic changes could not be demonstrated. The failure to demonstrate an abnormality in lymphocyte nucleotide incorporation during transformation suggests that the thiamine deficiency is limited to the myelocytic and erythroid series. Whether this patient's diabetes and deafness should be considered an integral part of the thiamine dependency is difficult to assess. Defective DNA synthesis, implied by the megaloblastic anemia, could also result in functional derangements of the pancreatic islet cells and of the cochlear nerve cells. The failure of the deafness and diabetes to respond to therapy does not necessarily indicate a lack of association with thiamine dependency. Therapy was not administered until years after the onset of symptoms, and "the disorders in these tissues may be irreversible. A role for thiamine in lysine and valine metabolism has not been described. Thiamine administration greatly altered postloading plasma lysine levels, implying a functional role for this vitamin in lysine metabolism. The abnormal urinary and plasma levels of valine after loading, however, was not changed by therapy. If the normal metabolism of this amino acid is in fact dependent upon thiamine, an irreversible lesion in this case must be postulated. One could postulate that this patient's disease involves a single defective enzyme requiring excessive thiamine concentrations for normal activity. In the presence of usual physiologic concentrations of the vitamin, this enzyme alone functions suboptimally and results in megaloblastic anemia. In contrast, a generalized thiamine deficiency alters the function of multiple thlamine-dependent enzymes and produces an entirely different clinical spectrum characterized as beriberi. Megaloblastic anemia is absent in beriberi, but this absence does not necessarily indicate that thiamine plays no role in blood formation. Rather, it suggests that lack of thiamine
Megaloblastic anemia
503
affects other metabolic processes more profoundly. Thus, a failing heart or a neurologic deficit may result in death before thiamine tissue concentrations fall to levels limiting hematopoiesis. Only when metabolic pathways most sensitive to thiamine deficiency are spared would the role of thiamine in blood formation be demonstrable. If a single thiamine-dependent enzyme is defective so that it alone requires increased concentrations of the vitamin, disordered hematopoiesis could occur in the absence of either thiamine deficiency or signs of beriberi. This postulated single-enzyme defect would, therefore, allow the demonstration of the heretofore unsuspected participation of thiamine in hematopoiesis. SUMMARY
An ll-year-old Caucasian girl is presented with a megaloblastic anemia, refractory to folate and vitamin B12, but responsive to treatment with thiamine. The initial response followed the administration of a multiple vitamin preparation. Withdrawal of supplemental vitamins resulted in a relapse within 3 ~ months. When the implicated vitamins were given sequentially, a reticulocytosis followed the administration of thiamine. A second relapse of the anemia occurred 4 months after cessation of vitamin supplementation. On this occasion the anemia was corrected by 20 rag. oral thiamine daily. The response of anemia to superphysiologic quantities of this vitamin supports a thiamine-dependent state and demonstrates a previously unsuspected participation of thiamine in hematopoiesis. Additional abnormalities include diabetes mellitus, aminoaciduria, and sensorineural deafness. The assay of urinary methylmalonic acid was performed by Frank A Oski, M.D., Hospital of the University of Pennsylvania, Philadelphia, Pa. The assay of urinary xanthurenie acid was performed by Dr. Ernest E. McCoy, University of Virginia School of Medicine, Charlottesville, Va. The amino acid analysis was performed courtesy of Dr. Paul Webster, Veterans Administration Hospital, Durham, N. C.
504
Rogers, Porter, and Sidbury
REFERENCES 1. Huguley, C. M., Jr., Bain, J. A., Rivers, S. L., and Seoggins, R. B.: Refractory m.egaloblastic anemia associated with excretion of orotic acid, Blood 14: 615, 1959. 2. MacGibbon, B. H., and Mollin, D. L.: Sideroblastic anaemia in man: Observations on seventy cases, Brit. J. Haemat. 11: 59, 1965. 3. Schwartz, S. O., and Critchlow, J.: Erythremic myelosis (di Guglielmo's disease). Critical review with report of four cases, and comments on erythroleukemia, Blood 7: 765, 1952. 4. Rogers, L. E., Warlord, L. R., Patterson, R. B., and Porter, F. S.: Hereditary orotic aciduria. I. A new case with family studies, Pediatrics 42: 415, 1968. 5. Rogers, L. E., and Porter, F. S.: Hereditary orotic aciduria. II. A urinary screening test, Pediatrics 42: 423, 1968. 6. Kohn, J., Mollin, D. L., and Rosenbach, L. M.: Conventional voltage electrophoresis for formiminoglutamic-acid determination in folic acid deficiency, J. Clin. Path. 14: 345, 1961. 7. Wolfe, S. J., Brin, M., and Davldson, C. S.: The effect of thiamine deficiency on human erythrocyte metabolism, J. Clin. Invest. 37: 1476, 1958. 8. Dreyfus, P. M., and Hauser, G.: The effect of thiamine deficiency on the pyruvate decarboxylase system of the central nervous system, Biochim. et biophys, acta 104: 78, 1965. 9. Haugen, H. N.: The determination of thiamine and thiamine phosphates in blood and tissues by the thiochrome method, Scandinav. J. Clin. & Lab. Invest. 13: 50, 1961. 10. Friedmann, T. E., and Kmieciak, T. C.: The determination of thiamine in blood, J. Lab. & Clin. Med. 28: 1262, 1963. 11. Eagle, H.: Nutrition needs of mammalian cells in tissue culture, Science 122: 501, 1955.
The Journal of Pediatrics April 1969
19. Moorhead, P. S., Nowell, P. C., Mellman, W. J., Battips, D. M., and Hungerford, D. A.: Chromosome preparations of leukocytes cultured from human peripheral blood, Exper. Cell Res. 20: 613, 1960. 13. Spackman, D. H., Stein, W. H., and Moore, S.: Automatic recording apparatus for use in chromatography of amino acids, Anal. Chem. 30: 1190, 1958. 14. Nyhan, W. L., Borden, M., and Childs, B.: Idiopathic hyperglycenemia: A new disorder of amino acid metabolism. II. The concentrations of other amino acids in the plasma and their modification by the administration of leucine , Pediatrics 27: 539, 1961. 15. Evered, D. F.: The excretion of amino acids by the human. A quantitative study with ion-exchange chromatography, Biochem. J. 62: 416, 1956. 16. Isra~ls, M. C. G., and Wilkinson, J. F.: Achrestic anaemia, Quart. J. Med. 5: 69, 1936. 17. Davidson, L. S. P.: Refractory megaloblastic anemia, Blood 3; 107, 1948. 18. Isra~ls, M. C. G., and Wilkinson, J. F.: New observations on the aetiology and prognosis of achrestic anaemia, Quart. J. Med. 9: 163, 1940. 19. Roelsen, E., and Ohlsen, A. S.: Achrestic anemia. Completely refractory megaloblastic anemia. A case followed for nearly four years, Acta reed. scandinav. 150: 17, 1954. 20. Brin, M.: Erythrocyte transketolase in early thiamine deficiency, Ann. New York Acad. Se. 98: 528, 1962. 21. Greengard, P.: In Goodman, L. S., and Gilman, A., editors: The pharmacological basis of therapeutics, ed. 3, New York, 1965, The Macmillian Company, p. 1655. 22. Sehreier, K.: Some peculiarities of amino acid metabolism in infancy and early childhood, J. P~DIAT. 46: 86, 1955.
April, 1969 T h e Journal o] P E D I A T R I C S
Thiamine-responsive megaloblastic anemia An 11-year-old Caucasian girl is presented who had a megaloblastic anemia responsive only to thiamine. Other abnormalities inekuded diabetes mellitus, aminoaciduria, and sensorineural dea[ness. Initially the anemia, re#actory to vitamin Ble and ]olic acid
therapy, responded to administration o] a multiple vitamin preparation. Vitamin supplementation was withdrawn ]ollowed by a recurrence o] anemia 3 ~ months later. The implicated vitamins were then administered sequentially. A retieuloeytosis [ollowed administration o] thiamine. Anemia again recurred 4 months a#er cessation o] vitamin supplementation. On this occasion the anemia was corrected by the oral administration o[ 20 rag. thiamine daily. Thiamine blood levels and activities o] 3 thiamine-dependent enzymes o] the patient's blood cells were normal, excluding a generalized de]eet o] thiamine metabolism. The patient there]ore appeared to have a thiamine-dependent megaloblastic anemia. This represents the first demonstration o] a role ]or this vitamin in D N A metabolism.
Lon E. Rogers, M.D., F. Stanley Porter, M.D.,* and James B. Sidbury, Jr., M.D. DURHAM~
N. G.
MEOALOBLASTIC A N E M IA S of childhood are rare and are most often the result of vitamin B12 or folic acid deficiency. R a r e r causes are hereditary orotic aciduria, 1 sideroblastic anemias, 2 and malignancies of the di G u g l i e l m o variety? This report presents a patient whose megaloblastic anemia was unresponsive to folic acid and vitamin B12 but responded to the administration of thiamine. From the Department o] Pediatrics, Duke University Medical Center. This work was supported by National Institutes o] Health Grants Nos. 5 TO1 H E 05478, 2 R O 1 A M 06815, and MO1 FR 30. Presented in part at the meeting o] the Society ]or Pediatric Research, Atlantic City, New Jersey, April 28, 1967. *Reprint requests: Department of Ped~atrlcs, Duke Un~verslty Medical Center, Durham, N. C. 27706 Vol. 74, No. 4, pp. 494-504
Anemia recurred twice during periods of normal dietary thiamine intake and was corrected by supplementation with the vitamin. This unique response of a megaloblastic anemia to thiamine led to additional studies of the patient, including the activities of known thiamine-dependent enzymes, blood thiamine levels, and the incorporation of nucleosides into lymphocyte nucleic acids.
CASE REPORT AND CLINICAL STUDIES The patient, an l l-year-old Caucasian girl, was born November 11, 1954, following an apparently normal gestation. The birth weight was 3.6 kilograms; growth and development during infancy were normal. The family history revealed no relatives with deafness, diabetes, or
Volume 74 Number 4
MegaIoblastic anemia
49 5
Fig. 2. Bone marrow showing megaloblasts.
Fig. 1. Peripheral blood smear showing macrocytosis, anisocytosis, and poikilocytosis. unexplained anemia. At 3 years of age the patient developed diabetes mellitus, and at 4 ~ years of age hearing loss was first noticed. Bilateral sensorineural deafness was complete by age 7I~ years. Hemoglobin determinations done at age 1, 7, and 10 7/12 years were 10.5, 11.5, and 10 Gm. per cent, respectively. In February, 1966, a severe anemia was first detected; the hemoglobin was 6.0 Gm. per cent. Oral iron was administered for 2 89 months without response, and a packed erythrocyte transfusion was then given. On April 5, 1966, a smear of a bone marrow aspirate revealed megaloblastic changes. A serum vitamin B12 level measured 756 /z/zg per milliliter (Bioscience Laboratories). Oral folic acid (5 rag. 3 times daily for 2 weeks) and parenteral vitamin B12 (100/Lg daily for 1 week) were administered without hematologic response. The patient was then referred for evaluation. Upon admission to Duke University Medical Center Hospital on June 28, 1968, the patient appeared pale and was obviously deaf. The weight was 32 kilograms, the height 134 cm. The tympanic membranes were normal in appearance. A soft precordial heroic murmur was audible; otherwise, the heart was normal. The liver, spleen, and lymph nodes were not palpably enlarged. There were no edema, peripheral neuropathies, or other signs of beriberi.
The results of the admitting laboratory work were as follows: hemoglobin 5.8 Gm. per cent, hematocrit 19 per cent, leukocytes 3,500 per cubic millimeter, platelets 145,000 per cubic millimeter, reticulocytes 4 per cent. The differential leukocyte count was: 4 bands, 49 polymorphonuclear forms, 32 lymphocytes, 11 monocytes, 4 eosinophils. The peripheral blood smear (Fig. 1) revealed moderate poikilocytosis and anisocytosis with numerous macrocytes, giant platelets, and a slight degree of nuclear hypersegmentation of the granulocytes. A smear of a bone marrow aspirate showed erythroid hyperplasia with numerous megaloblasts (Fig. 2). Little stainable iron and no ring sideroblasts were present. Hemoglobin electrophoresis revealed A hemoglobin. The sickle cell preparation was negative. Erythrocyte autohemolysis was 0.17 per cent at 24 hours, 0.84 per cent at 48 hours. Incubated erythrocyte osmotic fragility was normal. The serum iron concentration was 115 /~g per cent; the total iron binding capacity was 300/~g per cent. Beginning on July 3, 1966, Berocca-C, ~ 2 ml., was administered parenterally once daily for 5 consecutive days. A reticulocyte response of 10 per cent was first apparent on the fourth clay of treatment and reached a peak of 19 per cent on ~Berocca-C (Roche) contains per 2 ml.: thiamine HCI 10 mg., riboflavin I0 rag., pyrldoxine 20 rag., niacinamlde 80 rag., paathenol 20 mg., biotin 0.2 rag., aseorbie acid 100 mg.
49 6
Rogers, Porter, and Sidbury
the eighth day of treatment. The reticulocytosis
was followed by a rise in hemoglobin to a maximum of 9.8 Gin. per cent on July 19, 1966 (Fig. 3). At the same time the leukopenia and thrombocytopenia were corrected. A posttreatment bone marrow obtained on July 13, 1966, showed improvement but not complete resolution of the megaloblastic changes. Deca-Vi-Sol,t 1 chewable tablet daily, was administered from July 6 through July 17, 1966. On July 6, 1966 (after 3 days treatment with Berocca-C) the patient experienced transient hypoglycemia. The diabetes had been well controlled with an 1,800 calorie diet, NPH insulin 40 units, and regular insulin 90 units per day. During the following 10 days, however, hypoglycemic attacks necessitated a decrease of insulin dosage to approximately one half the pretreatment level. Supplemental vitamins were not administered after discharge from the hospital, and a balanced 1,800 calorie diabetic diet was continued unchanged. Follow-up laboratory tests on August 2, 1966, and August 29, 1966, revealed no change in the hematologic status. Blood smears continued to show moderate anisocytosis and poikilocytosis. By late August the insulin requirements had increased to 50 units N.P.H. insulin daily. Signs of hematologic relapse were present on Oct. 24, 1966, approximately 3~2 months after the first remission. The hemoglobin was 7.8 Gin. per cent, hematocrit 24 per cent, leukocyte count 5,400 per cubic millimeter, reticulocytes 1.8 per cent, platelets 107,500 per cubic millimeter, erythrocytes 2.34 • 106 per cubic millimeter, MCV 102 cubic microns, M C H 33 /~/xg, MCHC 33 per cent. The morphologic abnormalities of the peripheral blood smear were again present. The bone marrow was frankly megaloblastic. Stainable iron was present, but there were no ring sideroblasts. During the second admission to the hospital the vitamins present in Berocca-C were given in a sequential manner to ascertain which vitamin, if any, had" produced the original response. The vitamins given, the reticulocytosis, and the hemoglobin'response are shown in Fig. 4. A reticulocytosis followed the administration of thiamine. The reticulocyte count peaked at 22 per cent, 9 days after the initiation of thiamine, and was followed by a rise of hemoglobin to 13.6 Gin. "~Deca-Vi-Sol (Mead Johnson) contains per tablet: vitam i n A 4,000 units, vitamin D 400 units, ascorbic acid 75 rag., thiamine 1.2 rag., riboflavin 1.5 m g . , niacinamide 15 m g . , pyrldoxlne 1.2 m g . , pantothenlc acid 5 m g . , v i t a m i n B~s 3 /tg,
biotin 40 #g, ferrous fumarate 40 mg.
The Journal of Pediatrics April 1969
per cent. The borderline thrombocytopenia and leukopenia were also corrected, but insuliri requirements did not decrease as Previously. In an attempt to again induce improvement of the diabetes, biotin, pantothenic acid, and finally Berocca-C were administered. The insulin requirements did not change. Following the last injection of Berocca-C on Jan. 5, 1967, all supplemental vitamins were discontinued. No medication except insulin was taken. Hematologic remission continued for 4 months until May, 1967. Upon readmission in relapse, the hemoglobin was 8.5 Gin. per cent, erythrocytes 2.46 • 106 per cubic millimeter, leukocytes 6,000 per cubic millimeter, hematocrit 26 per cent, reticulocytes 6.3 per cent, platelets 180,000 per cubic millimeter, MCV 106 cubic microns, M C H 35/z/zg, MCHC 33 per cent. The bone marrow and peripheral blood smears showed the previously described abnormalities. Administration of oral uridine 1.0 Gin. per day for 5 days (May 11 to 15, 1967) and yeast DNA 1.5 Gm. per day for 5 days (May 16 to 20, 1967) failed to produce a hematologic response. Following these trials, thiamine, 20 rag. per day orally, was begun on May 22, 1967. Reticulocytosis began on the fourth day of treatment and peaked at 14.5 per cent on the eleventh day. The hemoglobin rose to 13.7 Gin. per cent by the twenty-eighth day of thiamine administration (Fig. 5). The leukocyte and platelet counts
- 2O
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Fig. 3. R e t i c u l o c y t o s i s and rise of h e m o g l o b i n foll o w i n g administration of a multiple vitamin preparation, Berocca-O.
Volume 74 Number 4
Megaloblastic anemia
were within the normal range during the second relapse and did not change with treatment. Following each of the hematologic responses, the abnormalities of the peripheral blood smear were not completely corrected; anisocytosis and poikilocytosis of erythrocytes persisted. In addition to the anemia, diabetes, and deafness, other abnormalities were present. An electroencephalogram obtained during hematologic relapse showed the following features: spike wave abnormalities of the temporal region of a 55~2 per second frequency and not exceeding 100 microvolts amplitude. In the parietal region, 6 per second high voltage discharges with amplitudes in the 200 microvolt range were seen. There was no alteration of this bizarre tracing with therapy. The patient had never had any seizure activity. Although her motor development was normal, her intellectual development was somewhat impaired. The deafness and unwillingness
to cooperate with simple instructions had made an exact evaluation of intelligence impossible, but her achievement at school was on a kindergarten level. Other laboratory findings included the following: serum sodium 136 mEq. per liter, potassium 5 mEq. per liter, carbon dioxide 28 mEq. per liter, calcium 9.8 mg. per cent, phosphorus 4.1 mg. per cent; blood urea nitrogen 9 mg. per cent; total serum protein 7.8 Gin. per cent, albumin 4.6 Gm. per cent, globulin 3.2 Gm. per cent, IgG 1,150 mg. per cent, IgA 100 mg. per cent, and IgM 59 mg. per cent, PBI 6.9 /xg per cent, serum glutamic-oxalacetic transaminase 15.4 units, serum glutamic-pyruvic transaminase 40 units, zinc turbidity 16 units, alkaline phosphatase 7.0 Bodanski units, and prothrombin time 83 per cent of normal. Routine urinalysis revealed: specific gravity 1.005, pH 6.5; the reactions for protein, sugar, and acetone were
I~- Thiomine----I Biotin Berocco-C - 24 Niocin I I I I I I Pontothenic Pantothenic 22 ir Acid Acid I I I I H
Vit. C I I P)~ridoxi~e 14
Riboflovin I I
2O
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18
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49 7
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Jan., ~67
Fig. 4. Reticulocytosis and rise of hemoglobin following administration of thiamine. Lack of response to pyridoxine, riboflavin, vitamin C, niacin, and pantothenic acid is shown. The daily dosages and dates of administration of the vitamins are as follows: pyridoxine 25 rag., Nov. 8-13, 1966; riboflavin 30 rag., Nov. 14-22; ascorbic acid 50 rag., Nov. 19-23; pantothenic acid 950 rag., Nov. 24-Dec. 2; niacin 50 rag., Nov. 30-Dec. 4; thiamlne 50 nag., Dec. 5-16; thiamine 95 mg., Dec. 17-22; biotin 10 rag., Dec. 19-16; pantothenic acid 63 rag., Dec. 20-22; Berocca-C 2 ml., Dec. 28-31; Berocca-C 1 ml., Jan. 1-5, 1967. All vitamins were given intramuscularly except biotin and riboflavin, which were taken orally.
4 98
Rogers, Porter, and Sidbury
The Journal of Pediatrics April 1969
,
15
,
15
14 13
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DNA I 16
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I I I I 3 5 ,June 1967
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19
Fig. 5. Reticulocytosis and rise of hemoglobin following administration of oral thiamine 20 mg. per day. Lack of response to uridine and DNA is shown.
negative; the microscopic examination revealed an occasional epithelial cell. Urinary FeCla, DNPH, and CTAB tests were negative. Additional studies of special interest included the following: Activities of the erythrocyte enzymes, orotidylic pyrophosphorylase, and orotidylic decarboxylase4 were determined and were not decreased. Urinary screening test for hereditary orotic aciduria 5 was also negative. Urinary formiminoglutamic acid excretion was not excessive after histidine load. 6 Urinary excretion of methylmalonic acid and xanthurenic acid during relapse was also normal. The diabetes was unresponsive to administration of tolbutamide. An electrocardio.gram during relapse was normal. Following the last remission the patient took 20 rag. thiamine orally per day and maintained a normal hemoglobin for more than one year. SPECIAL STUDIES The response of the megaloblastic anemia to thiamine suggested at least two possibilities regarding the underlying defect in this patient. The disorder might involve primarily the metabolism of thiamine and result
secondarily in the malfunction of numerous thiamine-dependent enzymatic processes , or the defect may lie in a specific thiaminedependent enzyme so that it alone operates suboptimally. To investigate these possibilities, the activities of 3 thiamine-dependent enzymes were assayed and blood thiamine levels were measured. T h e megaloblastic features of the anemia led to studies of the incorporation of uridine and thymidine by lymphocytes and of lymphocyte transformation following stimulation by phytohemagglutinin. The finding of elevated urinary amino acids led to their quantitation in urine and plasma. METHODS
Erythrocyte transketolase. The method used was that reported by Wolfe and associates. ~ Erythrocyte transketolase is assayed by cycling 2-C*4-glucose through the pentose shunt of the glycolytic pathway, a process requiring the activity of this thiamine-de-
Volume 74 Number 4
pendent enzyme. Loss of enzymatic activity is reflected by a deficient production of carbon dioxide-C ~* during the second transit of the sugar molecule through the pentose pathway. An erythrocyte suspension of known concentration was prepared from heparinized blood by centrifugation and by removal of plasma and buffy coat, followed by resuspension in an equal volume of buffer of p H 7.4 constituted as follows: 20 raM. sodium phosphate, 5 mM. magnesium chloride, 15 raM. sodium chloride, and 4 raM. potassium chloride. This buffer was also used in the other enzyme assays to be described. The reaction mixture consisted of a 0.4 ml. volume of the erythrocyte suspension and 0.2 ml. of aqueous 0.025 per cent methylene blue with 0.06/~c (5.06/~moles) 2-C 14glucose made up to a 1 ml. volume with buffer. Duplicate samples were run with and without thiamine hydrochloride in a final concentration of 10-s M. A blank was prepared by omitting the erythrocyte suspension. The samples were incubated at 37 ~ C. in a shaking water bath for 3 hours. The C ~4 02 was collected in hydroxide of Hyamine which was counted in a toluene-phosphor mixture in a Packard Tri-Carb Liquid Scintillation Counter. The results are expressed as mp.moles of COs per l0 s erythrocytes per hour. This and the following assays described were performed with blood taken from the patient during relapse. No supplemental thiamine was b e i n g administered. The enzymatic activities of control blood samples were determined simultaneously. Leukocyte pyruvlc dehydrogenase. Pyruvate is decarboxylated by leukocyte pyruvie dehydrogenase to form acetyl-CoA and carbon dioxide. The activity of this thiaminedependent enzyme was assayed by the recovery of carbon dioxide-C 14 following the incubation of leukocytes with 1-C~4-pyruvate. A modification of the method published by Dreyfus and HauseP was used. Cell suspensions were prepared by removal of plasma from heparinized whole blood following centrifugation. Since erythrocytes lack this enzyme, no attempt was made to remove them.
Megaloblastic anemia
499
Sedimented cells were resuspended in a volume of the buffer described above to provide a concentration of leukocytes from 5,000 to 10,000 per cubic millimeter (Coulter Counter). A volume of 0.7 ml. of this suspension was combined with 0.4 ml. of the buffer containing 0.017 ~c (72 /xmoles) 1-C14-sodium pyruvate. From this point the assay was performed with and without thiamine, as described for the erythrocYte transketolase assay. Results are expressed as m~moles CO2 per 105 leukocytes per hour. Leukocyte a-ketoglutaric dehydrogenase. The carboxyl carbon of a-ketoglutaric acid is released as CO2 upon the conversion of this acid to succinyl-CoA in the presence of oe-ketoglutaric dehydrogenase. The activity of this thiamine-dependent enzyme was assayed by the collection of carbon dioxide-C 14 when labeled o~-ketoglutaric acid was used as the substrate. Leukocytes were prepared and counted as previously described. A 0.5 ml. volume of the suspension was added to 0.5 ml. of buffer containing 0.08 ~c (6.35 /~moles) 1,2-C-~4-o~-ketoglutaric acid. From this point the assay was performed with and without thiamine, as described for the transketolase assay. Enzymatic activity of a normal control sample was determined simultaneously. The results are expressed as m/~moles CO2 per 105 leukocytes per hour. Blood thiamine assay: Blood thiamine was assayed according to the method of HaugenP Thiamine pyrophosphate was hydrolyzed with phosphatase to form the free vitamin which was oxidized by potassium ferricyanide to the fluorescent thiochrome. The fluorescence was measured after extraction of the thiochrome into isobutanol. Powdered Takadiastase (Parke-Davis) was used as a source of phosphatase? ~ All samples were measured in duplicate. Leukocyte transformation and incorporation studies. The basic medium was Eagle's M E M ~x with Earle's balanced salt solution. The p i t was adjusted to 7.4 with 5 per cent sodium bicarbonate. For the studies designed to demonstrate thiamine dependency, the medium was identical except that thiamine
500
Rogers, Porter, and Sidbury
was omitted in preparation. Phytohemagglutinin P was obtained from Difco Laboratories. Plasmagel was obtained from the Laboratorie Roger Bellon. Ha-thymidine and HS-uridine were obtained from New England Nuclear Corp. I-Ieparinized blood was obtained from the patient when in relapse and mixed with volume of Plasmagel for sedimentation of the red ceils. One milliliter of cells suspended in the plasma-Plasrnagel mixture was transferred to a stoppered test tube containing 4 ml. of culture medium containing peniciIlin and streptomycin as the antibiotics. Phytohemagglutinin P was added in a volume of 0.1 ml. Cultures were incubated at 37 ~ C. for 72 hours. Velban (Grand Island Biological Co.) was added two hours before harvesting to effect metaphase arrest. The cells were harvested and prepared after the method of Moorhead and associates. 12 In the studies with H3-thymidine and H 3uridine incorporation, 4 ~c of the tritiated nucleotide was added to the media 6 hours before harvesting. After the slides were fixed and stained, they were dipped in Kodak nuclear tract emulsion (NBT2) and kept in the Cambridge box for 6 weeks before being developed. Aminoacid studies. Aminoacid analyses were performed on aliquot parts of 24 hour urine collections. Plasma aminoacids were determined after precipitation of the protein with picric acid which was removed by passage through a column of Dowex 2 x 10. The aminoacids were separated and quantitated with a Beckman Spinco amino acid analyzer using the conditions described by Spackman and associates33 Loading icests were performed by oral administration of 150 mg. per kilogram of the amino acids lysine and valine; the tests were carried out before and during therapy with thiamine. Because of technical limitations only a 3 hour sample was obtained after loading, this being a time when the values would be expected to have returned to near control levels in the normal individual, but could be expected to be elevated if there were delayed clearance. 14
The Journal of Pediatrics April 1969
RESULTS Erythrocyte transketolase. Averaged values of duplicate determinations are expressed as m,amoles CO2 per l0 s erythrocytes per hour. Activities for the patient's erythrocyte transketolase were 12.0 with thiamine added to the reaction mix, and 11.9 without added thiamine. Corresponding activities for the control subject were 6.3 and 4.9, respectively. Leukocyte pyruvic dehydrogenase. Results are expressed as m~moles CO2 per 105 leukocytes per hour. Averaged values for activity of the pyruvic dehydrogenase for the patient were 1.6 with and 1.5 without added thiamine. For the control the respective values were 2.0 and 2.3. Leukocyte ~-ketoglutaric dehydrogenase. With enzyme activities expressed as m~moles CO2 per 10 5 leukocytes per hour, averaged values for duplicate samples for the patient were 0.45 with added thiamine and 0.29 without added thiamine. For the control the values were 0.51 with added thiamine and 0.38 without added thiamine. Blood thiamine assay. Values for duplicate samples of total blood thiamine for the patient during relapse averaged 4.8/~g per cent. Normal values by this method range from 2.48 to 10.77 ~g per cent with an average of 5.28 /~g per cent."
Leukocyte transformation and incorporation studies. The studies of lymphocyte transformation consequent to phytohemagglutinin stimulation were indistinguishable in the media with and without added thiamine in terms of the percentage of large lymphocytes and mitotic figures. No difference was demonstrated in tritiated thymidine or uridine incorporation with or without added thiamine in the media, as judged from the number of grains per cell. Normal control cells run simultaneously could not be distinguished from the patient's ceils by the response given. Amino acid studies. The urinary content of amino acids in a 24 hour specimen were: valine 61 mg. per 24 hours before therapy and 47 rag. per 24 hours during therapy with thiamine, compared with a normal range of 4 to 6 rag. per 24 hours; lysine 74 mg. per 24 hours before and 203 mg. per 24 hours
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Megaloblastie anemia
50 1
Table I. Plasma amino acids (milligrams per 100 ml.). The quantitative results of plasma aminoacids in the control period and 3 hours after valine and lysine loading (150 rag. per kilogram) are presented. The loading tests were run before therapy was instituted and several days after high doses of thiamine had been administered. Average adult control values are listed for comparison ~2
Amino acid Aspartic acid Threonine Serine Proline Glutamic acid Glycine Alanine Half eystine Methionine Isoleueine Leucine Tyroslne Phenylalanine Histidine Arginine Lyslne Valine
Fasting Trace 0.76 0.78 0.99 0.81 1.20 1.35 Trace Trace Trace 0.81 Trace Trace 1.35 Trace 1.26 1.46
Valine load Lysine load Be[ore thiAfter thiBe[ore thiAlter thiamine therapy amine therapy amine therapy amine therapy 0.40 Trace 0.09 Trace 3.35 1.49 1.99 2.29 3.41 L72 1.89 1.69 2.7O 0 4.62 0 4.16 1.04 2.43 1.10 3.48 1.67 4.24 1.40 7.97 4.29 8.44 4.9 0 Trace Trace Trace 1.06 0.55 Trace 0.37 2.98 1.76 0.22 1.46 7.37 1.98 1.69 1.99 0 1.2 0 1.07 0 0.83 0 0.69 3.52 1.30 1.01 1.65 2.56 Trace Trace 0.91 20 4.36 3.98 1.68 38.16 27.81 9.75 3.2
during therapy, compared with normal range of 7 to 48 mg. per 24 hours. 15 The other amino acids were present in normal quantities. The finding of elevated valine and lysine levels in the urine prompted the investigation of the plasma levels and loading response to these 2 amino acids. The plasma sample was obtained before and 3 hours after the load to discriminate abnormal clearance of the loaded dose from the normal. 1~ The values obtained with the valine loading were abnormal and were the same before and after thiamine therapy. Lysine clearance was definitely abnormal before, but normal after, therapy was instituted. There was a small but significant rise in proline with both loading tests before therapy, and a decrease to undetectable levels in response to these loading tests after therapy was commenced (Table I ) . DISCUSSION
All of the usual causes of megaloblastic anemia may be excluded in this patient. .Classical pernicious anemia is excluded by
Average adult control ee
0.5 2.0 1.4 2.6 1.1 2.8 3.8 1.0 0.35 1.6 2.0 1.4 1.6 1.7 2.1 2.8 3.0
the normal serum vitamin BI~ level, the failure of response to parenteral vitamin B~, and the normal urinary excretion of methylmalonie acid. Folic acid deficiency is similarly excluded by the lack of response to folate therapy and by the normal urinary excretion of formiminoglutamic acid. di Guglielmo's erythremic myelosis is a progressive, malignant process unresponsive to known hematinics and usually terminates fatally within months? Hereditary orotic aeiduria is excluded by assays of the activities of the enzymes, orotidylic pyrophosphorylase and orotidylic decarboxylase, and by the urinary screening test for this disorder. 4' ~ Megaloblastic changes are aIso found in the bone marrow of some patients with sideroblastic anemia. Signs of folate deficiency and a partial response to this vitamin or to pyridoxine are sometimes observed. Characteristic findings are increased numbers of ringed sideroblasts in the marrow and an excessive excretion of urinary xanthurenie acid. The patient's anemia, on the other hand, was unresponsive to both pyridoxine and folic acid,
5 0 2 Rogers, Porter, and Sidbury
the bone marrow was not iron loaded, and the urinary excretion of xanthurenic acid and formiminoglutamic acid was not elevated. Isolated cases of megaloblastic anemia totally unresponsive to therapy or respondi n g to unusual therapeutic regimens have been reported. 16-19 These disorders, however, do not present the same features as the patient reported here. The response of this patient's anemia to the administration of thiamine is unique and suggests one of several possible derangements of thiamine metabolism: deficient intake, defective absorption, excessive excretion, inadequate utilization, or an increased requirement. The first three possibilities may be excluded for the following reasons: (1) Clinical signs of beriberi were not present, (2) blood thiamine levels were normal, (3) erythrocyte transketolase activity was not reduced. The transketolase assay is an especially sensitive indicator of thiamine deficiency, and low blood levels of this vitamin for any reason (decreased intake, inadequate absorption, or excessive excretion) would be expected to result in decreased activity, s~ Furthermore, anemia, if present at all, is a late manifestation of thiamine deprivation. The presence of a severe anemia without any other signs of beriberi is not readily explained on the basis of a generalized thiamine deficiency. Thiamine pyrophosphate is a cofactor for several essential enzymatic reactions such as the transketolation reaction and the decarboxylation of o~-ketoacids. In an attempt to exclude a generalized failure of thiamine utilization in this patient, 3 thiamine-dependent enzymes were assayed: erythrocyte transketolase, leukocyte pyruvic dehydrogenase, and leukocyte a-ketoglutaric dehydrogenase. The activities of these particular enzymes were found to be normal, and their activities were not significantly altered by the addition of thiamine hydrochloride in vitro. The finding of 3 normally functioning thiamine-dependent enzymes in this patient's blood cells demonstrates that the defect does not affect all enzymes utilizing this vitamin co factor.
The ]ournal o[ Pediatrics April 1969
The most reasonable explanation for the response of this child's anemia to thiamine is an increased requirement for this vitamin, a thiamine-dependent state. Whereas the ingestion of normal dietary amounts of thiamine was insufficient to prevent the development of anemia, it was corrected by the administration of amounts significantly greater than the accepted minimum daily requirement. This finding suggests that normal hematopoiesis is made possible in this child by the elevation of tissue thiamine concentrations. High thiamine intake may load tissues and expand stores 2 to 3 times normal, el but once supplementation is withdrawn, a prompt decrease of stores to pretreatment level is to be expected. In the studies of Brin, 2~ for example, human volunteers were given 5 mg. thiamine daily for 5 days, an amount adequate to replete all stores, and thereafter thiamine-poor diets were ingested. Biochemical evidence of thiamine deficiency was apparent 8 days later, and symptoms of depletion appeared within 21 days. Apparently so long as this patient's tissue level of thiamine could be maintained by supplementation at superphysiologic levels, normal hematopoiesis was possible. With physiologic levels of tissue thiamine, however, hematopoiesis was retarded and within weeks resulted in anemia. The response of the disorder to large but not to physiologic amounts of the vitamin has a parallel in pyridoxineresponsive anemia. 2 The megaloblastic features of the anemia suggest that the postulated defect involves the synthesis of nuclear material (DNA). Until now there has been no evidence to implicate thiamine in DNA synthesis. To study this possibility further, DNA and uridine were administered to the patient. In addition, lymphocytes obtained during hematologic relapse were incubated with labeled thymidine and uridine and examined autoradiographically. The administration of DNA and uridine failed to produce a hematologic response, and the patient's lymphocytes incorporated thymidine and uridine normally. In addition, Iymphocytes s t i m u 1 a t e d with
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phytohemagglutinin underwent characteristic blast transformation. From these studies the mechanism of impairment of nucleic acid metabolism accounting for the megaloblastic changes could not be demonstrated. The failure to demonstrate an abnormality in lymphocyte nucleotide incorporation during transformation suggests that the thiamine deficiency is limited to the myelocytic and erythroid series. Whether this patient's diabetes and deafness should be considered an integral part of the thiamine dependency is difficult to assess. Defective DNA synthesis, implied by the megaloblastic anemia, could also result in functional derangements of the pancreatic islet cells and of the cochlear nerve cells. The failure of the deafness and diabetes to respond to therapy does not necessarily indicate a lack of association with thiamine dependency. Therapy was not administered until years after the onset of symptoms, and "the disorders in these tissues may be irreversible. A role for thiamine in lysine and valine metabolism has not been described. Thiamine administration greatly altered postloading plasma lysine levels, implying a functional role for this vitamin in lysine metabolism. The abnormal urinary and plasma levels of valine after loading, however, was not changed by therapy. If the normal metabolism of this amino acid is in fact dependent upon thiamine, an irreversible lesion in this case must be postulated. One could postulate that this patient's disease involves a single defective enzyme requiring excessive thiamine concentrations for normal activity. In the presence of usual physiologic concentrations of the vitamin, this enzyme alone functions suboptimally and results in megaloblastic anemia. In contrast, a generalized thiamine deficiency alters the function of multiple thlamine-dependent enzymes and produces an entirely different clinical spectrum characterized as beriberi. Megaloblastic anemia is absent in beriberi, but this absence does not necessarily indicate that thiamine plays no role in blood formation. Rather, it suggests that lack of thiamine
Megaloblastic anemia
503
affects other metabolic processes more profoundly. Thus, a failing heart or a neurologic deficit may result in death before thiamine tissue concentrations fall to levels limiting hematopoiesis. Only when metabolic pathways most sensitive to thiamine deficiency are spared would the role of thiamine in blood formation be demonstrable. If a single thiamine-dependent enzyme is defective so that it alone requires increased concentrations of the vitamin, disordered hematopoiesis could occur in the absence of either thiamine deficiency or signs of beriberi. This postulated single-enzyme defect would, therefore, allow the demonstration of the heretofore unsuspected participation of thiamine in hematopoiesis. SUMMARY
An ll-year-old Caucasian girl is presented with a megaloblastic anemia, refractory to folate and vitamin B12, but responsive to treatment with thiamine. The initial response followed the administration of a multiple vitamin preparation. Withdrawal of supplemental vitamins resulted in a relapse within 3 ~ months. When the implicated vitamins were given sequentially, a reticulocytosis followed the administration of thiamine. A second relapse of the anemia occurred 4 months after cessation of vitamin supplementation. On this occasion the anemia was corrected by 20 rag. oral thiamine daily. The response of anemia to superphysiologic quantities of this vitamin supports a thiamine-dependent state and demonstrates a previously unsuspected participation of thiamine in hematopoiesis. Additional abnormalities include diabetes mellitus, aminoaciduria, and sensorineural deafness. The assay of urinary methylmalonic acid was performed by Frank A Oski, M.D., Hospital of the University of Pennsylvania, Philadelphia, Pa. The assay of urinary xanthurenie acid was performed by Dr. Ernest E. McCoy, University of Virginia School of Medicine, Charlottesville, Va. The amino acid analysis was performed courtesy of Dr. Paul Webster, Veterans Administration Hospital, Durham, N. C.
504
Rogers, Porter, and Sidbury
REFERENCES 1. Huguley, C. M., Jr., Bain, J. A., Rivers, S. L., and Seoggins, R. B.: Refractory m.egaloblastic anemia associated with excretion of orotic acid, Blood 14: 615, 1959. 2. MacGibbon, B. H., and Mollin, D. L.: Sideroblastic anaemia in man: Observations on seventy cases, Brit. J. Haemat. 11: 59, 1965. 3. Schwartz, S. O., and Critchlow, J.: Erythremic myelosis (di Guglielmo's disease). Critical review with report of four cases, and comments on erythroleukemia, Blood 7: 765, 1952. 4. Rogers, L. E., Warlord, L. R., Patterson, R. B., and Porter, F. S.: Hereditary orotic aciduria. I. A new case with family studies, Pediatrics 42: 415, 1968. 5. Rogers, L. E., and Porter, F. S.: Hereditary orotic aciduria. II. A urinary screening test, Pediatrics 42: 423, 1968. 6. Kohn, J., Mollin, D. L., and Rosenbach, L. M.: Conventional voltage electrophoresis for formiminoglutamic-acid determination in folic acid deficiency, J. Clin. Path. 14: 345, 1961. 7. Wolfe, S. J., Brin, M., and Davldson, C. S.: The effect of thiamine deficiency on human erythrocyte metabolism, J. Clin. Invest. 37: 1476, 1958. 8. Dreyfus, P. M., and Hauser, G.: The effect of thiamine deficiency on the pyruvate decarboxylase system of the central nervous system, Biochim. et biophys, acta 104: 78, 1965. 9. Haugen, H. N.: The determination of thiamine and thiamine phosphates in blood and tissues by the thiochrome method, Scandinav. J. Clin. & Lab. Invest. 13: 50, 1961. 10. Friedmann, T. E., and Kmieciak, T. C.: The determination of thiamine in blood, J. Lab. & Clin. Med. 28: 1262, 1963. 11. Eagle, H.: Nutrition needs of mammalian cells in tissue culture, Science 122: 501, 1955.
The Journal of Pediatrics April 1969
19. Moorhead, P. S., Nowell, P. C., Mellman, W. J., Battips, D. M., and Hungerford, D. A.: Chromosome preparations of leukocytes cultured from human peripheral blood, Exper. Cell Res. 20: 613, 1960. 13. Spackman, D. H., Stein, W. H., and Moore, S.: Automatic recording apparatus for use in chromatography of amino acids, Anal. Chem. 30: 1190, 1958. 14. Nyhan, W. L., Borden, M., and Childs, B.: Idiopathic hyperglycenemia: A new disorder of amino acid metabolism. II. The concentrations of other amino acids in the plasma and their modification by the administration of leucine , Pediatrics 27: 539, 1961. 15. Evered, D. F.: The excretion of amino acids by the human. A quantitative study with ion-exchange chromatography, Biochem. J. 62: 416, 1956. 16. Isra~ls, M. C. G., and Wilkinson, J. F.: Achrestic anaemia, Quart. J. Med. 5: 69, 1936. 17. Davidson, L. S. P.: Refractory megaloblastic anemia, Blood 3; 107, 1948. 18. Isra~ls, M. C. G., and Wilkinson, J. F.: New observations on the aetiology and prognosis of achrestic anaemia, Quart. J. Med. 9: 163, 1940. 19. Roelsen, E., and Ohlsen, A. S.: Achrestic anemia. Completely refractory megaloblastic anemia. A case followed for nearly four years, Acta reed. scandinav. 150: 17, 1954. 20. Brin, M.: Erythrocyte transketolase in early thiamine deficiency, Ann. New York Acad. Se. 98: 528, 1962. 21. Greengard, P.: In Goodman, L. S., and Gilman, A., editors: The pharmacological basis of therapeutics, ed. 3, New York, 1965, The Macmillian Company, p. 1655. 22. Sehreier, K.: Some peculiarities of amino acid metabolism in infancy and early childhood, J. P~DIAT. 46: 86, 1955.