Pigmentation in Megaloblastic Anaemia Associated with Pregnancy and Lactation

Generalized skin pigmentation in five African women with megaloblastic anaemia in the postnatal period was associated with low serum folate levels, as distinct from vitamin B₁₂ deficiency. It is suggested that the occurrence of pigmentation in both folate and vitamin B₁₂ deficiency may reflect a common abnormality of metabolism.     

Base Composition of Normal and Megaloblastic Bone Marrow DNA

PARRY has speculated that in megaloblastic anaemia, deficiency of folate or vitamin B₁₂ may mean that a population of cells is formed in the marrow containing DNA with subnormal amounts of thymidine, so accounting for some of the abnormalities of megaloblastic erythropoiesis¹. We have therefore determined the base composition of DNA from normal and megaloblastic cells and have also measured the amounts of 5-methylcytosine^2,3 present in these DNA preparations, as vitamin B₁₂ and folate are known to be concerned in many methylation reactions in man⁴ and could be directly or indirectly concerned in the formation of 5-methylcytosine.     

Nutritional Anemia and Megaloblastosis in Pregnancy

Macrogranulocytic and/or erythroid megaloblastic bone marrow changes which could not be accurately predicted from the hematologic findings in the blood were present in 25% of 305 mildly to moderately anemic pregnant women attending a public antepartum clinic in Montreal. Iron deficiency was the primary cause of anemia in most instances. Serum folate activity of less than 4.1 ng./ml. and/or serum vitamin B₁₂ levels of less than 100 pg./ml. were present in 90% of the 77 patients having these bone marrow changes, whereas approximately one-third of 228 patients with normoblastic marrow had these low values. Red cell folate did not correlate as well as serum folate activity with bone marrow changes. After treatment with oral folic acid in the range of 0.2 mg. to 0.8 mg., daily, for seven to 14 days, the megaloblastic and macrogranulocytic changes in patients with low serum folate activity and normal serum vitamin B₁₂ values disappeared in 15 of 21 patients. Of five women having both low folate and vitamin B₁₂ values, three failed to respond and two showed only partial improvement after 0.4 mg. of folic acid daily, per os, for 10 days. The average diet of these anemic women was suboptimal in folate and in iron.     

Assessment of Deoxyuridine Suppression Test in Diagnosis of Vitamin B12 or Folate Deficiency

Deoxyuridine (dU) suppression tests have been performed on virtually all marrow samples aspirated at this hospital over the past 12 months. Of the 110 samples studied 26 gave abnormal results, and these 26 samples came from patients deficient in either vitamin B₁₂ or folate. The dU suppression test was found to be of particular value in the diagnosis of vitamin B₁₂ or folate deficiency in non-anaemic patients with macrocytosis and equivocal changes in marrow morphology and in patients in whom the serum vitamin B₁₂ or red cell folate levels were within the normal range.     

Serum Folate and Vitamine B12 Levels in Acute and Chronic Renal Disease. Effect of Peritoneal Dialysis

Serum folate and vitamin B₁₂ levels have been measured in 32 patients with renal failure. The initial mean serum folate level was raised above normal in seven patients with acute renal failure whereas the mean level in eight patients severely ill from chronic renal failure was significantly lower than normal. Serum folate levels fell during peritoneal dialysis and rose between dialyses in all these patients and also in one patient who was dialysed for acute pancreatitis.The mean serum B₁₂ level was raised in patients with both acute and chronic renal failure, but there was no consistent change in serum B₁₂ level during dialysis.Hypersegmented polymorphs were present in the peripheral blood film of most of the patients with acute or chronic renal failure. Their presence bore no relation to the clinical state, blood urea, serum folate, or serum B₁₂ level of the patients.     

The development and role of international biological reference materials in the diagnosis of anaemia

Anaemia is a major global health problem. Although the main cause is iron deficiency, anaemia also results from other nutritional deficiencies (folate and vitamin B₁₂), haemolytic disorders including haemoglobinopathies, and bone marrow disorders. Accurate diagnosis of anaemia is dependent on reliable diagnostic tests and reference ranges, which in turn are dependent on effective standardisation. Standardisation is achieved through the availability of reference materials and reference measurement procedures. International biological reference materials have therefore been developed to standardise and control diagnostic tests for anaemia for a diverse range of analytes including total haemoglobin and haemoglobin types, ferritin, the serum transferrin receptor, serum vitamin B₁₂ and folate, whole blood folate, and alloantibodies which mediate immune haemolytic anaemia.     

Malabsorption in Overland Travellers to India

Thirty-four cases of malabsorption are described in young adults after brief periods of overland travel to India. Symptoms included diarrhoea, abdominal distension, and weight loss. Investigation revealed fat, xylose, and vitamin B₁₂ malabsorption with marked morphological changes in the mucosa. Lower levels of serum folate and vitamin B₁₂ were observed in those with protracted diarrhoea, but no anaemia developed. Malabsorption may persist for many months after return to the U.K. Most patients responded initially to antibiotics, but some subsequently relapsed. The reasons why these patients developed tropical sprue are discussed.     

The Incidence and Prevention of Folate Deficiency in a Pregnant Clinic Population

Non-anemic women attending a public antenatal clinic were given, daily, a multivitamin tablet containing 78 mg. of elemental iron. The follow-up studies included an analysis of their diets. A total of 311 patients were included, of which one group received a supplement of 0.5 mg. folic acid and 0.005 mg. vitamin B₁₂. The incidence of megaloblastic bone marrow change in the unsupplemented group was 26% and of low blood folates approximately 50%. The incidence of megaloblastic changes was sharply reduced in the supplemented group and the blood folates were elevated to supranormal levels, indicating that the dose of folic acid used may have been above the minimal requirement. Formiminoglutamic acid (FIGLU) excretion could not be correlated with other parameters of folate deficiency. Neutrophil lobe counts did not relate to megaloblastic changes or low folate levels unless there was more than 5% hypersegmentation. The dietary intake was suboptimal in total calories, iron and food folate.     

Vitamin B12 Malabsorption after Cobalt Teletherapy for Carcinoma of the Bladder

After cobalt teletherapy for carcinoma of the bladder, eight out of 14 consecutively admitted patients were shown to have malabsorption of vitamin B₁₂, though none had developed a megaloblastic anaemia. Despite lack of symptoms this group of patients is at risk after radiotherapy.     

Free and Total Vitamin B12 in Cerebrospinal Fluid

Free and total vitamin B₁₂ levels in serum and cerebrospinal fluid (CSF) were bioassayed, since there were no available data on the relationship between free and total vitamin B₁₂ in CSF or between free vitamin in serum and CSF vitamin B₁₂. The subjects were 43 neurological patients. Serum levels were normal in 40 of 43 patients. Values for free and total vitamin B₁₂ in CSF were the same in 42 of 43 patients. Mean CSF vitamin B₁₂ was 21 μμg./ml. In 17 cases CSF vitamin B₁₂ equalled free vitamin B₁₂ level in serum, in 16 cases CSF vitamin B₁₂ was lower than the free level in serum, and in 10 cases CSF vitamin B₁₂ was higher than the free vitamin level in serum. There was no apparent diagnostic correlation. The findings suggest that vitamin B₁₂ is not bound in CSF and that there is some selective control of passage of vitamin B₁₂ across the blood-CSF barrier.     

Megaloblastic Anemia Associated with Surgically Produced Gastrointestinal Abnormalities

Two of the mechanisms for vitamin B₁₂ deficiency, leading to megaloblastic anemia, are the result of surgically produced abnormalities of the gastrointestinal tract. The basic mechanism is different for each lesion.Total gastrectomy results in complete lack of intrinsic factor which is necessary for vitamin B₁₂ absorption. It is believed that if patients survive long enough and are not given prophylactic vitamin B₁₂ therapy, all would develop megaloblastic anemia.Intestinal anastomosis leading to stasis of intestinal contents, with overgrowth of bacteria may cause vitamin B₁₂ deficiency through bacterial interference with the utilization of vitamin B₁₂.Use of radioactive vitamin B₁₂ (cobalt⁶⁰-labeled B₁₂) has led to a better understanding of the pathogenesis of both types of megaloblastic anemia. The radioactive vitamin provides a useful tool for study of its absorption from the gastrointestinal tract.     

Oxidation of compounds metabolized through folate coenzyme pathways in vitamin B12-deficient rats

The effects of vitamin B₁₂ deficiency in rats and dietary supplementation with vitamin B₁₂ and/or l-methionine plus folate on the oxidation of compounds metabolized through folate coenzyme pathways were investigated. Rats fed a vitamin B₁₂-deficient diet oxidized significantly lower amounts in 60 min of l-histidine, glycine, sarcosine, formate, and l-serine to CO₂ than vitamin B₁₂-supplemented controls. Supplementation of the deficient diet with l-methionine plus folate restored the ability to oxidize the ring-2-carbon of l-histidine, the methyl group of sarcosine, and formate to the same level as that observed in animals receiving vitamin B₁₂. In contrast, oxidation of the 1-carbon of glycine and the 3-carbon of l-serine was not restored to control levels by addition of methionine plus folate to the vitamin B₁₂-deficient diet. Inhibition of the metabolism of the 2-carbon of glycine to CO₂ was partially overcome by additional dietary methionine and folate. Glycine synthase activity in homogenates paralleled the in vivo pattern of oxidation of the 1-carbon of glycine to CO₂, whereas sarcosine dehydrogenase activity appeared to increase 2-fold in vitamin B₁₂ deficiency.     

Serum Vitamin B12 Levels and Vitamin B12 Binding Capacity in Pregnant and Non-pregnant Europeans and West Indians

The vitamin B₁₂ level and the capacity of serum to bind B₁₂ are higher in the West Indian population living in Great Britain than in Europeans. The B₁₂ level fell during pregnancy in both groups but remained higher in the West Indians. West Indians had higher levels of IgG.     

Value of Estimating Methylmalonic Acid Excretion in Anaemia

A rapid technique suitable for routine pathology laboratories has been used to estimate methylmalonic acid excretion in a 24-hour urine collection following a 10g. valine load. Levels above 40 mg./24 hours were found only in patients with vitamin B₁₂ deficiency. Patients with pernicious anaemia treated more than 24 hours before urine collection and patients with other types of anaemia had methylmalonic acid levels below 25 mg./24 hours.This method of demonstrating vitamin B₁₂ deficiency can be applied rapidly in debilitated patients so that specific treatment can be instituted within 36 hours of admission.     

Correlation of Serum and Urine Vitamin B12

The relation between the serum vitamin B₁₂ level and the daily loss of vitamin B₁₂ in urine was examined in patients with normal serum vitamin B₁₂ levels and in patients suffering from vitamin B₁₂ deficiency. A linear correlation was found between the two measurements, suggesting that the serum vitamin B₁₂ level is a governing factor in the urinary loss of vitamin B₁₂. The contribution by this loss to the total loss of vitamin B₁₂ from the body is small under normal circumstances but becomes quantitatively more important with the depletion of body stores.     

Higher Prevalence of Metformin-Induced Vitamin B12 Deficiency in Sulfonylurea Combination Compared with Insulin Combination in Patients with Type 2 Diabetes: A Cross-Sectional Study

Long-term and high-dose treatment with metformin is known to be associated with vitamin B₁₂ deficiency in patients with type 2 diabetes. We investigated whether the prevalence of B₁₂ deficiency was different in patients treated with different combination of hypoglycemic agents with metformin during the same time period. A total of 394 patients with type 2 diabetes treated with metformin and sulfonylurea (S+M group, n = 299) or metformin and insulin (I+M group, n = 95) were consecutively recruited. The vitamin B₁₂ and folate levels were quantified using the chemiluminescent enzyme immunoassay. Vitamin B₁₂ deficiency was defined as vitamin B₁₂≤300 pg/mL without folate deficiency (folate>4 ng/mL). The mean age of and duration of diabetes in the subjects were 59.4±10.5 years and 12.2±6.7 years, respectively. The mean vitamin B₁₂ level of the total population was 638.0±279.6 pg/mL. The mean serum B₁₂ levels were significantly lower in the S+M group compared with the I+M group (600.0±266.5 vs. 757.7±287.6 pg/mL, P<0.001). The prevalence of vitamin B₁₂ deficiency in the metformin-treated patients was significantly higher in the S+M group compared with the I+M group (17.4% vs. 4.2%, P = 0.001). After adjustment for various factors, such as age, sex, diabetic duration, duration or daily dose of metformin, diabetic complications, and presence of anemia, sulfonylurea use was a significant independent risk factor for B₁₂ deficiency (OR = 4.74, 95% CI 1.41–15.99, P = 0.012). In conclusion, our study demonstrated that patients with type 2 diabetes who were treated with metformin combined with sulfonylurea require clinical attention for vitamin B₁₂ deficiency and regular monitoring of their vitamin B₁₂ levels.     

Changes in the tissue concentration of folate and vitamin B12 during maturation and ageing

Age-dependent changes in folate and vitamin B₁₂ metabolism of mice have been investigated. The concentration of folate in liver plasma and blood showed a postnatal increase to a maximum at approx. 25 weeks. Total folate concentrations then remained constant whereas free folate decreased slowly up to week 98. Conversely both total and free folate of the brain were reduced extensively during the first 10 weeks of life after which time total folate concentration stabilised whilst that of free folate continued to decline slowly. The concentration of vitamin B₁₂ in brain, liver and plasma showed an initial rapid increase. The vitamin continued to accumulate more slowly in the brain and liver from weeks 10 to 98. The concentration of vitamin B₁₂ in the plasma appeared to achieve equilibrium after a period of accumulation lasting 25 weeks. These results suggest that during maturation the characteristics of folate metabolism of the brain are distinct from those of peripheral tissues, and that folate, unlike vitamin B₁₂ metabolism, undergoes continuing change with advancing age.     

Malabsorption of protein bound vitamin B12.

Patients with subnormal serum vitamin B12 concentrations were tested for absorption of protein bound vitamin B12 and compared with controls. Absorption of the protein bound vitamin appeared to decrease with increasing age in healthy subjects. Differences between the result of this test and the result of the Schilling test in patients who had undergone gastric surgery were confirmed; such differences were also seen in some patients who had iron deficiency anaemia, an excessive alcohol intake, or folate deficiency. Defective absorption was also found in six patients with an adequate dietary intake of vitamin B12, normal Schilling test results, low serum vitamin concentrations, and tissue changes responding to treatment with vitamin B12. Malabsorption of the vitamin from protein bound sources, which is not detected by the Schilling test, may produce vitamin B12 deficiency of clinical importance.     

Defective DNA synthesis in megaloblastic anaemia. Studies employing velocity sedimentation in alkaline sucrose gradients.

1. Autoradiographic experiments revealed that the average size of the replicating unit (replicon) in human phytohaemagglutunin-stimulated lymphocytes is 45 (+/- 1.3) micron. 2. A 5 min pulse of [3H]thymidine labelled DNA chains of approximately 40 S (15 micron) in control lymphocytes as revealed by velocity sedimentation in alkaline sucrose density gradients. Upon chasing in the absence of [3H]-thymidine the labelled DNA increased in size. By 6 h the bulk of the label co-sedimented with full-sized chromosomal DNA. 3. In untreated lymphocytes from patients with megaloblastic anaemia due to vitamin B-12 or folate deficiency or lymphocytes treated with methotrexate (10(-5) M) or hydroxyurea (5 . 10(-4) M) the increase in size of pulse-labelled DNA was slower than in control cells. 4. The block in maturation of pulse-labelled DNA to bulk DNA was not permanent. At 24 h of chase 75-80% of the pulse-label in both control and megaloblastic lymphocytes co-sedimented with bulk DNA. 5. We conclude that the lesions seen in DNA synthesis in megaloblastic anaemia due to folate or vitamin B-12 deficiencies occur through impaired biosynthesis of nucleotide precursors of DNA. Possible explanations of why the defects in DNA synthesis cause altered morphology of proliferating cells in megaloblastic anaemia are suggested.     

THE EFFECT OF VITAMIN B12 ON THE ANEMIA AND COMBINED SYSTEM DISEASE OF ADDISONIAN PERNICIOUS ANEMIA

The effect of the parenteral administration of vitamin B₁₂ has been observed in eight patients with Addisonian pernicious anemia.Vitamin B₁₂ in initial doses of 50 micrograms or 25 micrograms induced satisfactory reticulocyte response and a return of erythrocyte count to within normal range in 60 days.In only two of the patients were secondary reticulocyte responses induced on a second injection of vitamin B₁₂.Concurrently with the hemopoietic response, the bone marrow changed from megaloblastic hyperplasia to normoblastic distribution.The paresthesias associated with combined system disease as well as disturbances in position sense and locomotor function may be entirely relieved or greatly diminished following injections of vitamin B₁₂.Maintenance injections of vitamin B₁₂ may be from 30 to 50 micrograms at intervals of one month, the amount depending upon the individual case.Vitamin B₁₂ may be used without untoward symptoms in patients previously sensitive to liver extract.