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A female child with deafness was diagnosed to have neonatal diabetes mellitus at the age of 6 months, on routine evaluation prior to cochlear implant surgery. She presented to us at 11 months of age with diabetic ketoacidosis due to an intercurrent febrile illness. Her haematological parameters showed megaloblastic anaemia and thrombocytopenia. Therefore a possibility of Thiamine Responsive Megaloblastic Anaemia (TRMA) syndrome was considered. She was empirically treated with parenteral thiamine hydrochloride (Hcl). Subsequently, due to the unavailability of pharmacological preparation of oral thiamine Hcl in a recommended dose she was treated with benfotiamine. She had a sustained improvement in all her haematological parameters on oral benfotiamine. The insulin requirement progressively reduced and she is currently in remission for last 2 years. The genetic analysis confirmed the diagnosis of TRMA syndrome. Thus benfotiamine can be considered a new treatment option in management of TRMA syndrome.

Vitamin B(12) deficiency in vegans is a known cause of megaloblastic anaemia. We report an adolescent girl who presented with jaundice and weight loss for 6 months secondary to vitamin B(12) deficiency, leading to megaloblastic anaemia. Replacement with vitamin B(12) reversed her symptoms, resulting in weight gain, and normalised her haemoglobin, red blood cell morphology, bilirubin levels and serum vitamin B(12) levels.

Methylation of cytosine residues in DNA samples, collected before and serially after cobalamin treatment from patients with cobalamin deficiency, was studied using restriction endonucleases Hpa II and Msp I and an epsilon globin gene probe. There was no evidence of hypomethylation in any of the samples. It was concluded that although hypomethylation of metabolites such as choline occurs, that of DNA is preserved in megaloblastic anaemia.

Owing to the higher serum cobalamin results that are obtained by R-binder radioisotopic dilution assay compared to microbiological assays (E. gracilis and L. leichmannii) it was suggested that serum contained a cobamide(s) that could not be detected by the more specific microbiological assays and that a much less specific test organism, which responds to most naturally occurring cobamides, such as the cobamide-dependent E. coli mutant, might respond to these cobamide(s) in serum. In an attempt to investigate this possibility an improved and simplified E. coli assay for the measurement of cobamide in serum was developed. The method is described, and the results obtained in normal subjects, in patients with megaloblastic anemia, and in anaemic pregnant women not suffering from megaloblastic anaemia are reported and compared with E. gracilis assay results.

Impaired absorption of vitamin B12 has been shown by a whole body counter technique in 7 of 10 infants and children who had resection of over 45 cm of ileum. Absorption was normal in 2 patients who had 15 cm or more terminal ileum remaining. If absorption was abnormal the serum level of vitamin B12 did not fall below the lower limit of the normal range for several years, puberty being a particularly vulnerable time. Though the site and length of the resection often suggested whether vitamin B12 deficiency was likely to occur, an absorption test was necessary for accurate prediction and to allow treatment to be given rationally before complications occurred. The whole body counter technique was particularly suitable for children as it did not involve collection of urine or faeces, injections, or admission to hospital.

Subnormal red-cell folate values were encountered in 20 of 63 thalassaemic subjects in a population with a low incidence of megaloblastosis. The abnormality was not associated with haematological changes of megaloblastosis and could not be explained on the basis of incomplete liberation of intra-erythrocytic folates, serum conjugase deficiency of vitamin B12 deficiency. Evidence is presented to indicate that it is unlikely to represent a state of subclinical folate deficiency. The exact cause of the abnormality is unknown.

The definitive diagnosis of pernicious anaemia (PA) in the elderly is by no means always straightforward, particularly when inappropriate medication has been introduced before the institution of specific investigatory procedures. A detailed haematological study was carried out on 301 patients aged 60-95 with a serum B 12 concentration at the laboratory9s lower level of normal of 150 ng per litre ( Euglena gracilis assay). The diagnosis of PA was based on strict predetermined haematological criteria. All patients were subsequently studied by the simultaneous oral administration of the dual isotopes 57 Co-labelled B 12 bound to intrinsic factor and free 58 Co-labelled B 12 (Dicopac test), and urine was collected over 24 hours after an intramuscular dose of 1 mg nonradioactive B 12 for estimation of the 57 Co/ 58 Co B 12 ratio; 255 patients satisfied all criteria for final analysis. The Radiochemical Centre, Amersham suggests an upper limit of the normal range for the 57 Co/ 58 Co ratio of 1·3 with a lower limit for PA of 2·0. We were unable to show a sharp borderline in the 57 Co/ 58 Co B 12 ratio between those patients shown by other criteria to have PA and those who do not have PA; 34% of the 71 established patients had a ratio below 2·0. From our series a ratio borderline drawn at 1·4 gave only one false negative (1·4% of the PA group). Of the 175 non-PA cases, nine (5%) gave false positive results; four of these had 58 Co excretion levels high enough to make misdiagnosis unlikely. In a proportion of patients the 57 Co/ 58 Co B 12 ratio was estimated at regular intervals for 36-hour periods. Maximum accuracy of isotope measurement on a single specimen was obtained 8-20 hours after isotope dosing. The Dicopac investigation is a useful simple screening test in the differential diagnosis of patients with a megaloblastic bone marrow and combined low serum B 12 and folate concentrations. When carried out by the standard technique, the degree of discrimination between normal and abnormal ratios is of limited diagnostic significance in one-third of patients.

Only two (2.5%) of 80 outpatients with histologically proven ulcerative colitis had folate deficiency associated with anaemia or macrocytosis. Mean folate absorption, measured using micrograms/kg body weight of a tritium-labelled physiological folate derivative, 5-methyltetrahydroteroylglutamic acid, in six newly diagnosed patients was 76.7% (normal greater than 95%) but fell to 69.4% after three months' treatment with sulphasalazine. Mean difference in individual patients was 7.5% +/- 5.2% (SD) (p less than 0.02). Mean folate absorption in four patients with megaloblastic anaemia or macrocytosis which developed during treatment with sulphasalazine was 66.3%. This rose to 82.4% after the drug was stopped. Mean difference in individual patients was 16.6 +/- 6.6% (SD) (p less than 0.001). All patients who developed anaemia or macrocytosis with sulphasalazine had additional reasons for folate deficiency. These included coeliac disease, severe nutritional deficiencies, and haemolysis. It was concluded that sulphasalazine impairs folate absorption but this only becomes significant if other reasons for folate deficiency are also present.

The activity of the two folate dependent enzymes supplying carbon 2 and carbon 8 of the purine nucleus was assayed in the bone marrow of rats and man, as well as in rat livers. The activity of both enzymes was several fold greater in marrow than in liver. Inactivation of cobalamin by exposure to nitrous oxide did not affect the enzymes in rat marrow cells, although an appreciable effect on hepatic enzymes was found. The depression of hepatic glycinamide ribotide (GAR) transformylase in rats exposed to nitrous oxide was prevented by supplying a formate precursor, methylthioadenosine. There was a considerable rise in the activity of GAR transformylase in human marrow cells from patients with megaloblastic anaemia due to cobalamin deficiency but no change in activity in marrow from patients deficient in folate.

In 21 patients with megaloblastic anaemia associated with the tropical malabsorption syndrome serum vitamin B 12 levels have been measured serially before and during parenteral folic acid therapy. In 11 patients there was a significant rise in serum B 12 levels, reaching a peak three to 17 days after starting the folic acid. In three of these cases, absorption of labelled B 12 was the same before and after the administration of folic acid. In two cases there was a fall in serum B 12 levels and in seven there was no change. In one case small amounts of folic acid produced no change in serum B 12 levels, whereas larger doses produced a prompt but transient rise.

The routine examination of buffy coat films is a valuable diagnostic aid to screening large numbers of specimens of blood and is applicable to hospital and general practice, provided that a bottle of sequestrinated venous blood can be examined in the laboratory within 24 hours.The diagnosis of folic-acid deficiency anaemia of pregnancy can be made, in many cases, before the appearance of megaloblasts in the peripheral blood by finding macrocytes, polylobing of polymorphonuclear leucocytes, and ;out of step' haemoglobinization of normoblasts in the buffy coat film. Accurate haemoglobin estimations at intervals throughout pregnancy, and particularly in the last three months, are an important and in some cases essential part of ante-natal care. Any haemoglobin found to be below 75% (11.1 g.%) should be followed by the examination of a series of buffy coat films while the anaemia is being treated.

of serum levels of vitamin B12 and folate has meant that megalo blastic anaemias can be more readily classified as being due to deficiency of one or occasionally both of these substances. The excretion of formiminoglutamic acid (figlu) in the urine of patients with folic-acid deficiency has further aided this differen tiation. However, unless these tests clearly indicate deficiency of one substance with normal levels of the other, differentiation is often not precise. It has been shown that in vitamin-B12 deficiency serum-folate levels can be low, normal, or raised (Hansen and Weinfeld, 1962), and in folic-acid deficiency the vitamin-B12 level may be normal or reduced (Johnson et al., 1962. Figlu excretion may also occur in patients with vitamin B12 deficiency (Chanarin et al., 1962). There would therefore seem to be a place for the use of a therapeutic trial of vitamin

There is a significant fall in the serum folic acid level during pregnancy, reaching its lowest level at term. This is most pronounced in twin pregnancies. A similar but less spectacular fall occurs in the vitamin B(12) concentration. In megaloblastic anaemia both folic acid and vitamin B(12) levels are lower than in other pregnant women. The degree of megaloblastic change in the bone marrow, as measured by the type and number of megaloblasts, is reflected in the vitamin levels, cases with florid megaloblastosis showing the most marked depression of vitamin B(12) and folic acid activity. Although there is a significant difference in the mean folic acid levels between megaloblastic and normoblastic pregnant women, a considerable overlap exists between individual values in the two groups. When the labile folic-acid factor is determined separately the test becomes much more specific. In the present series, all cases of megaloblastic anaemia yielded labile-factor levels below 1.0 mmug. per ml., while a similar value was encountered in only one of 35 normal pregnancies. In five women with megaloblastic anaemia the vitamin B(12) concentration was less than 100 mumug. per ml. but rose to normal levels on folic acid therapy alone.

We report a case of congenital isolated malabsorption of folic acid, the first in a boy. Only seven previous cases have been reported, and we discuss two aspects--namely, the tendency to infection, with evidence of impairment of both cellular and humoral immunity, and the absence of neurological disturbances.

Ten patients with severe megaloblastic anaemia were studied to investigate whether the causative metabolic defects might predispose them to lactic or other acidosis. One patient had compensated acidosis with hyperlactataemia before treatment but there were obvious causes other than anaemia. No other patient developed an acidosis. Neither anaemia per se nor the metabolic defects of vitamin B(12) or folic acid deficiency are likely to cause clinically significant lactic acidosis or hyperlactataemia.

Urinary amino-acid chromatograms from 23 patients with megaloblastic anaemia have been studied before and after therapy. The most consistent abnormality was an increased taurine or increased taurine/glycine ratio. This was not related directly to deficiency of vitamin B(12) or folic acid or to the degree of anaemia.

Folic acid deficiency with the picture of a megaloblastic bone marrow may develop in haemolytic anaemia, and, on the other hand, both vitamin B(12) and folic acid deficiency may produce signs of haemolysis. As the correct interpretation of a positive antiglobulin reaction associated with megaloblastic erythropoiesis is particularly important, the effect of deficiency of vitamin B(12) and folic acid on the results of the test was investigated in 32 patients with vitamin B(12) or folic acid deficiency and a positive antiglobulin reaction was obtained in ten. There was no correlation between the result of the test and the degree of anaemia, and there was no significant difference between the incidence of positive results associated with deficiency of vitamin B(12) or folic acid. In determining the significance of a positive result, the time interval before agglutination occurs is sometimes of greater value than the strength of the reaction or the result of the gamma globulin neutralization test.