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The administration of vitamin E, a natural antioxidant, to a patient with paroxysmal nocturnal haemoglobinuria (PNH) failed to diminish the urinary excretion of 59-Fe as monitored by 59-Fe whole body counting and urinary loss of isotope. However, in vitro vitamin E corrected the increased sensitivity of PNH red cells to haemolysis by hydrogen peroxide. These results support the concept that the susceptibility of PNH red cells to lipid peroxidation is an in vitro phenomenon bearing little relation to the mechanism of in vivo haemolysis.

Folate is essential for DNA synthesis and the survival and growth of the malaria parasite. Folate sufficiency may be associated with an increased risk of malaria. Antifolate antimalarial drugs are of major importance in the prophylaxis and treatment of malaria. Folic acid reverses the inhibition by antifolate drugs of plasmodial growth or survival in vitro, and folic acid supplements given to children with malaria may increase the failure rate of treatment with antimalarials. There is no convincing evidence of a significant prevalence of folate deficiency in children in malarious areas, nor of a beneficial effect of folic acid supplementation on malarial anemia. In areas where Plasmodium falciparum malaria is holoendemic, universal supplementation of children with iron and folic acid may increase the incidence of severe morbidity and mortality. These regions should be excluded from the World Health Organization recommendation of universal folic acid supplementation of children in areas of high prevalence of anemia. This does not apply to supplementation of pregnant women with folic acid.

We are researching and writing a book on my late aunt, Dr Mary Gordon. When she immigrated to South Africa (SA) from England in 1917, she became the first woman doctor to be appointed to Johannesburg Hospital. She was later to establish a large private practice in Johannesburg while also teaching at the then newly founded medical school at the University of the Witwatersrand, where she remained one of the members of staff until 1946.

Background: Limited blood supplies necessitate the rational use of blood products. The aim of the present study was to provide a basis for audits of red cell usage in surgery by benchmarking common practice. Application of the data to the construction of a maximum surgical blood order schedule may be relevant for centres that perform a serological crossmatch or who collect autologous units. Methods: Data on surgical procedures identified by Commonwealth Medical Benefits Schedule item numbers, were collected retro­spectively from theatre and blood bank records at the Royal Melbourne and Melbourne Private hospitals from May 1997 to April 1998. The percentage of procedures for which red cells were transfused, and the mean, median and range of units transfused for procedures with ≥ 30% transfusion likelihood were identified. Results: Over 12 months, 266 surgical procedure codes were itemized ≥ 10 times each, contributing 12 300 data entries. Only 38 procedures demonstrated an incidence of transfusion of at least 30%. Most frequently transfused procedures included spinal fusion, total hip replacement, mandible/maxilla resection, prostatectomy and bladder excision. Conclusion: The number of common surgical procedures in which there is a 30% or greater likelihood that red cell transfusions will be given is limited. This benchmarking of common red cell usage is a first step in the process of determination of transfusion appropriateness.

Background: The aim of the present paper was to review the pattern of collection and transfusion of autologous red cells for elective surgical procedures Methods: Data on requests for preoperative autologous donation of blood were obtained from the Australian Red Cross Blood Service, Victoria and the Royal Melbourne Hospital for the calendar year 1998. The following information was collected: patient age, sex, surgery type, number of autologous units requested and collected and, if relevant, reasons for not achieving the requested collection. Transfusion of autologous units and any additional homologous units was confirmed from records at the blood banks of the Royal Melbourne Hospital and Melbourne Pathology (the pathology provider performing cross-matching for the majority of autologous units collected by Australian Red Cross Blood Service, Victoria). Results: Over 12 months, 2803 units of autologous blood were requested and 2282 units collected from 1301 patients. The most common reason for failure to collect the number of units requested was insufficient time between referral and surgery. Of the autologous units collected, 73% were transfused giving a collection to transfusion ratio of 1.4. Sixty-eight per cent of patients received their autologous units only, 10% received both autologous and homologous units, while 22% were not transfused. For the majority of procedures, patients using preoperative autologous donation of blood had higher transfusion rates than those who did not use this. Conclusions: Ninety per cent of patients undergoing preoperative autologous donation of blood successfully avoided homologous blood exposure. However, preoperative autologous donation of blood is both wasteful and increases the incidence of transfusion in surgical procedures.

OBJECTIVE To determine the effect of prospective monitoring on appropriateness of transfusions of red cells, platelets and fresh frozen plasma (FFP). DESIGN Prospective interventional study. SETTING Royal Melbourne Hospital (a tertiary teaching hospital), Melbourne, Victoria, March-May 1996. INTERVENTION The blood product request form was modified to incorporate indications for transfusion and clinical and laboratory data. Requests were monitored by blood bank laboratory staff for conformation with hospital transfusion guidelines; non-conforming requests were discussed with the requesting medical practitioner by the Haematology Registrar before blood products were issued. In case of disagreement, blood products were always issued. SUBJECTS 200 consecutive transfusion episodes for each product (red cells, platelets and FFP). OUTCOME MEASURES Appropriateness of transfusion, assessed by a Consultant Haematologist according to hospital guidelines. Rates of inappropriate transfusion episodes after intervention were compared with rates in a previous study. RESULTS After intervention, rates of inappropriate transfusion episodes fell significantly (red cells, 16% to 3% [P = 0.004]; platelets, 13% to 2.5% [P = 0.02]; and FFP, 31% to 15% [P = 0.02]). Almost all inappropriate FFP transfusion episodes post-intervention were due to failure to demonstrate prolongation of prothrombin or activated partial thromboplastin times more than 1.5 times the control value. CONCLUSION Prospective monitoring of request forms can reduce rates of inappropriate transfusions. High rates of inappropriate FFP transfusions possibly reflect uncertainty about appropriate laboratory criteria for FFP transfusion. While results of large prospective randomised controlled clinical trials of FFP transfusions are awaited, currently laboratory criteria can be retained, but should be applied with flexibility.

The purpose of this study was to determine if there is an increased prevalence of the thrombophilic genetic mutant factor V Leiden in patients with recurrent miscarriage. Functional assays were conducted for activated protein C resistance and genetic detection of factor V Leiden in 100 women with recurrent miscarriage compared with a control group. The prevalence of factor V Leiden in patients was 6/100 (6%) compared with 3/85 (3.5%) in controls. The difference was not statistically significant. Antithrombotic prophylaxis with heparin and/ or aspirin in a subsequent pregnancy in five of the six patients with factor V Leiden was associated with maintenance of pregnancy and delivery of a live, healthy, full-term infant in four. We have been unable to demonstrate a statistically significant increased prevalence of factor V Leiden in women with recurrent miscarriage. If antithrombotic prophylaxis in pregnancy can be shown by controlled therapeutic trial to prevent miscarriage in these patients, identification of this subgroup would be important. Key Words: Recurrent miscarriage—Factor V Leiden—Activated protein C resistance.

OBJECTIVES: To determine the clinical significance of subnormal serum vitamin B12 concentration in older people by comparing the hematological, neurological, and biochemical findings in patients with subnormal serum B12 with a control group with normal B12 levels. DESIGN: Clinical and laboratory assessment of hospital patients selected to represent a wide range of serum B12 levels. SETTING: Patients in the medical wards of two hospitals, one a general hospital and the other a geriatric hospital. PARTICIPANTS: Ninety-four older patients, 43 with subnormal (

Objective To determine how current hospital practice for transfusions of red cells, platelets and fresh frozen plasma conformed with published criteria. Design Elaboration of criteria for transfusion from a review of the current literature; and analysis of the medical records of patients receiving transfusions of red cells (200), platelets (215), and fresh frozen plasma (260) during defined time periods in 1993. Setting A large tertiary care teaching hospital. Outcome measures Inappropriateness rates for transfusion episodes and numbers of individual units of blood products administered. Results Inappropriateness rates for transfusion episodes and numbers of individual units administered were 16% and 10% for red cells, 13% and 11% for platelets, and 24% and 16% for fresh frozen plasma (31% and 21% when transfusions for thrombotic thrombocytopenic purpura were excluded). Red cells and fresh frozen plasma were used inappropriately most frequently in association with a surgical procedure; for platelets, it was their use for bleeding. In many of the transfusions deemed inappropriate, deficiencies of red cells, platelets and/or coagulation factors were documented, but the degree of deficiency did not meet the stringent appropriateness criteria. Twenty-six transfusions were deemed inappropriate because the indication was not documented in the medical record. Conclusions Specific problem areas in which blood product use was inappropriate were identified. Guidelines for transfusion appropriateness, education of hospital staff, and a monitoring system to ensure adherence to the guidelines, are required.

A full blood count which shows anaemia and macrocytosis should prompt the practitioner to look for

Based on biochemical evidence, a high prevalence of biochemical evidence of vitamin B12 or folate deficiency has been reported in a number of areas in the world. The evidence that these biochemical abnormalities lead to a comparable prevalence of anemia is reviewed. The overall contribution of vitamin B12 deficiency to the global burden of anemia is probably not significant, except perhaps in women and their infants and children in vegetarian communities. In developed countries, folate-deficiency anemia is uncommon. In some developing countries, this anemia is still seen, but there are no comprehensive data on the relative prevalence compared with anemia due to malaria, iron-deficiency, hemoglobinopathy, and HIV disease. It seems unlikely that folate deficiency makes a major contribution to the burden of anemia in developing countries. Iron-deficiency anemia may coexist with vitamin B12 and especially folate deficiency, and may confound the hematological features of the vitamin deficiencies whose prevalence would then be underestimated. Supplementation of the diet of pregnant women with folic acid can virtually eliminate folate-deficiency anemia in these women. There are very few data on the hematological effect of vitamin B12 supplementation or fortification at the population level. The addition of vitamin B12 to the supplementation of the diet of pregnant women with iron and folic acid does not produce an increased hematological response, at least in nonvegetarian populations. There are numerous reports of the effect of folic acid fortification of food on tests of folate status, but only a single published report on the hematological response was found.

Limited blood supplies necessitate the rational use of blood products. The aim of the present study was to provide a basis for audits of red cell usage in surgery by benchmarking common practice. Application of the data to the construction of a maximum surgical blood order schedule may be relevant for centres that perform a serological crossmatch or who collect autologous units.Data on surgical procedures identified by Commonwealth Medical Benefits Schedule item numbers, were collected retrospectively from theatre and blood bank records at the Royal Melbourne and Melbourne Private hospitals from May 1997 to April 1998. The percentage of procedures for which red cells were transfused, and the mean, median and range of units transfused for procedures with/= 30% transfusion likelihood were identified.Over 12 months, 266 surgical procedure codes were itemized/= 10 times each, contributing 12 300 data entries. Only 38 procedures demonstrated an incidence of transfusion of at least 30%. Most frequently transfused procedures included spinal fusion, total hip replacement, mandible/maxilla resection, prostatectomy and bladder excision.The number of common surgical procedures in which there is a 30% or greater likelihood that red cell transfusions will be given is limited. This benchmarking of common red cell usage is a first step in the process of determination of transfusion appropriateness.

The aim of the present paper was to review the pattern of collection and transfusion of autologous red cells for elective surgical proceduresData on requests for preoperative autologous donation of blood were obtained from the Australian Red Cross Blood Service, Victoria and the Royal Melbourne Hospital for the calendar year 1998. The following information was collected: patient age, sex, surgery type, number of autologous units requested and collected and, if relevant, reasons for not achieving the requested collection. Transfusion of autologous units and any additional homologous units was confirmed from records at the blood banks of the Royal Melbourne Hospital and Melbourne Pathology (the pathology provider performing cross-matching for the majority of autologous units collected by Australian Red Cross Blood Service, Victoria).Over 12 months, 2803 units of autologous blood were requested and 2282 units collected from 1301 patients. The most common reason for failure to collect the number of units requested was insufficient time between referral and surgery. Of the autologous units collected, 73% were transfused giving a collection to transfusion ratio of 1.4. Sixty-eight per cent of patients received their autologous units only, 10% received both autologous and homologous units, while 22% were not transfused. For the majority of procedures, patients using preoperative autologous donation of blood had higher transfusion rates than those who did not use this.Ninety per cent of patients undergoing preoperative autologous donation of blood successfully avoided homologous blood exposure. However, preoperative autologous donation of blood is both wasteful and increases the incidence of transfusion in surgical procedures.

Objectives To determine the effect of small doses of oral cyanocobalamin supplements in older patients with low or borderline serum vitamin B12 concentrations but no other evidence of pernicious anemia (PA). Design Randomized, double-blind, placebo-controlled study assessing the efficacy of oral cyanocobalamin 10 microg and 50 microg daily for 1 month. Setting Two geriatric hospitals in the North Western Health Care Network, Melbourne, Australia. Participants Thirty-one inpatients with serum vitamin B12 levels between 100 and 150 pmol/L, without PA, other malabsorption disorders, or progressive neurological or terminal illness. The mean age was 81.4 years. Intervention After informed consent, a medical and drug history was taken and the Mini-Mental State Examination (MMSE) completed. A dietitian made assessment of oral cobalamin intake. Blood was taken for serum vitamin B12, serum and red cell folate assay, full blood examination, fasting serum gastrin, parietal and intrinsic factor antibodies, fasting serum homocysteine, and creatinine. Patients were then randomized to receive 10 microg oral cyanocobalamin, 50 microg oral cyanocobalamin, or placebo treatment for 1 month, after which the investigations and clinical examinations were repeated. Measurements Percentage change in the level of vitamin B12, homocysteine, folate, and red cell parameters and absolute changes in MMSE were calculated and compared between groups. The groups were compared on the number of responders who improved their level of B12 by 20%. Chi-square calculations on changes in serum vitamin B12 concentration were also performed. Results Mean serum vitamin B12 +/- standard deviation improved by 51.7 +/- 47.1% in the 50-microg group, 40.2 +/- 34.4% in the 10-microg group, and 11.7 +/- 24.5% in the placebo group. The change in the 50-microg cyanocobalamin group was significantly greater than that in the placebo group (P=.044). The change in the 10-microg cyanocobalamin group was not significantly different from that in the placebo group (P=.186). Eight of 10 subjects in each treatment group were classified as responders, compared with two of 11 in the placebo group (P=.004). Homocysteine levels fell in patients receiving cyanocobalamin, but this fall failed to reach statistical significance. There were no significant changes in the other parameters measured. Conclusion Cyanocobalamin supplementation of 50 microg but not 10 microg daily produced a significant increase in serum vitamin B12. This result has implications for the management of patients with subnormal or borderline serum vitamin B12 concentrations and for food fortification with vitamin B12.

Reports from some Western countries indicate that mandatory folate food fortification (FFF) has substantially reduced the prevalence of folate deficiency, leading to calls for folate testing following FFF to be limited to specific indications such as macrocytic anaemia. This is premature for low-income countries, where folate deficiency is predominantly the result of poor intake coupled with the increasing demand in pregnancy. There is also evidence that HIV infection is prejudicial to folate nutrition, and low-income HIV-infected women and their offspring could be among the most susceptible to folate deficiency. In assessing folate nutrition, the value of serum folate has been compromised by FFF, and both serum and red cell folate are necessary for optimal assessment of folate status. Although the limited data available suggest that large-scale masking of vitamin B12 deficiency by FFF has not occurred, it has been suggested that B12 be incorporated into folate-fortified foods. However, significant B12 deficiency is usually due to malabsorption, and physiological doses added to food would be of questionable value because they would not be absorbed. Extensive work, especially randomised clinical trials, must be done before dietary intervention with B12 on a national scale can be justified.

To determine the significance of the commonly observed fall in serum vitamin B12 levels during pregnancy, serum levels of the B12 metabolites methylmalonic acid (MMA) and homocysteine (Hcy) were measured in a group of 50 pregnant patients with subnormal serum B12 (range 45-199 pg/ml) and the results compared with those of 25 pregnant controls (serum B12208-580 pg/ml). Mean values for serum MMA and total Hcy in the subnormal B12 group were 445.4 nmol/L and 7.03 μmol/L, respectively, which were not significantly different from the mean MMA of 440.5 nmol/L and Hcy of 6.88 nmol/L in the controls. For the total group of patients, neither serum MMA nor serum Hcy levels correlated with serum B12. One-third of pregnant patients showed elevated serum MMA values, independent of B12 status. Significant elevation of serum Hcy was detected in only two patients, both with subnormal serum B12 and hematological evidence of B12 deficiency. We conclude that the usual fall in serum B12 concentration in pregnancy does not reflect B12 deficiency at the biochemical level. In establishing true B12 deficiency in pregnancy, the serum Hcy level (in the absence of folate deficiency) but not serum MMA, is of value. © 1995 Wiley-Liss, Inc.

Myeloid sarcoma is a neoplasm of immature myeloid cells occuring in an extramedullary site. It rarely occurs in the testis and if so, is almost always associated with recurrent, co-existing or is the initial presentation of bone-marrow based disease. This is usually acute myeloid leukaemia and less commonly, myelodysplastic or myeloproliferative disorders. We describe a case of myeloid sarcoma as an isolated mass in the testis of a seventy-six year old; without past or current history of acute myeloid leukaemia. We discuss the histologic, cytochemical and immunohistochemical findings that brought us to the diagnosis. The differential diagnoses considered in this case include plasmacytoma, diffuse large B-cell lymphoma, anaplastic large cell lymphoma, precursor lymphoblastic lymphoma/leukaemia and metastatic amelanocytic melanoma. The literature was reviewed in order to calculate the range as well as mean interval time between first presentation of myeloid sarcoma and acute myeloid leukaemia and to calculate how long cases defined as myeloid sarcoma isolated to testis had been followed-up. Our aim is to determine how long such a patient should be followed-up for in order to accurately diagnose the entity of myeloid sarcoma confined to testis without bone-based disease.

Idiopathic hypereosinophilic syndrome (HES) comprises a heterogeneous group of disorders characterised by prolonged eosinophilia with no obvious cause. A patient with longstanding HES is reported in whom unusual non-neoplastic peritrabecular lymphoid aggregates were present in the bone marrow, a hitherto undescribed association, as far as is known. An eosinophil colony stimulating activity was detected in the serum. The findings in this patient provide further evidence for an important role for eosinophil colony stimulating activity interleukin-5 mediated T lymphocyte control of eosinophil production in the pathogenesis of the HES.

Low levels of cerebrospinal S-adenosylmethionine in association with abnormal myelination in inherited disorders affecting cobalarnin and folate metabolism provide new data on a possible mechanism of cobalarnin neuropathy.

Cobalamin neuropathy was produced in Cape fruit bats (Rousettus aegyptiacus) by a cobalamin-free diet combined with intermittent exposure to nitrous oxide, which inactivates cobalamin. Cobalamin-deficient bats had low hepatic methylmalonyl-CoA mutase holoenzyme activity, with elevated plasma and urinary methylmalonic acid levels. No significant changes could be demonstrated in the concentration of odd- or branched-chain fatty acids in the nervous system. Supplementation of the cobalamin-free diet with methionine, valine or isoleucine delayed the onset of neuropathy, despite persistence of methylmalonic acid accumulation. Supplementation with any of the three amino acids was associated with elevation of hepatic methionine concentration. The action of valine and isoleucine in delaying the onset of neuropathy can be explained by their methionine-sparing effect. These results emphasize the central role of methionine in the cobalamin neuropathy and do not support the hypothesis that the neuropathy is related to an effect of cobalamin deficiency on the propionic acid pathway.

o|1. Vitamin B 12 deficiency induced in the fruit bat by a combination of dietary deprivation and exposure to nitrous oxide (N 2 O) is accompanied by profound neurological impairment, thus providing an experimental model for the study of vitamin B 12 neuropathy. 2. Electron microscopy of the spinal cord of vitamin B 12 deficient bats shows marked changes in the myelin of the posterior columns in the form of distension, separation and vacuolation of myelin lamellae similar to the changes described in the dietary induced B 12 deficient monkey model. 3. No equivalent change occurred in bats exposed to N 2 O and supplemented with vitamin B 12 .

1. The effect of cobalamin inactivation by the anaesthetic gas nitrous oxide on the concentration of S-adenosylmethionine (Ado Met) in brain and liver of fruit bats (Rousettus aegyptiacus) was examined.2. Test animals exposed to N2O–oxygen (50:50, v/v) developed ataxia and paralysis leading to death after an average of 9·8 weeks (n 6). Animals receiving pteroylmonoglutamic acid supplements in the diet became ataxic earlier (mean 8·8 weeks) while those receiving methionine supplements survived for significantly longer periods (12·5 weeks, P < 0·01).3. Plasma cobalamin levels indicated severe depletion of cobalamin stores in N2O-exposed animals.4. The mean concentration of Ado Met in the brain of N2O-treated bats was nearly 50% higher than that of untreated controls. Ado Met levels in treated bats receiving pteroylmonoglutamic acid or methionine supplements were respectively 18 and 25% higher than in controls. In contrast, the concentration of Ado Met in the liver of all the N2O-treated groups was slightly lower than in controls.5. These results suggest that the N2O-induced neuropathy in the fruit bat is not related to a depletion of Ado Met in the nervous system.

The effects of vitamin B-12 deficiency on lipids and fatty acids of spinal cord myelin were studied in control and vitamin B-12-deficient fruit bats. Very low plasma and brain vitamin B-12 concentrations were present in animals fed the vitamin B-12-free, all fruit diet. Myelin was isolated from the spinal cord of control and vitamin B-12-deficient animals (n = 3 pools) by means of a flotation method in a discontinuous sucrose gradient. The molar concentration of cholineglycerophosphatide was significantly lower in the deficient bats than in the controls. The molar cholesterol: phospholipid ratio in the deficient bats was also significantly lower. The odd-chain fatty acid 15:1 was not detected in cholineglycerophosphatide from the controls, but comprised 1.4% of the total fatty acids in the deficient group. The odd-chain fatty acids 15:0, 17:1 and 19:0 were present in slightly higher amounts in cholineglycerophosphatide of deficient bats. Lipid and fatty acid differences were related to the vitamin B-12-requiring methionine synthetase and methylmalonyl CoA mutase reactions.

In 1969 a group of hunter-gatherer San were studied (Am J Clin Nutr 1971;24:229-42). Their state of hematological nutrition was excellent with a negligible incidence of iron, folate, or vitamin B12 deficiency. A genetically and linguistically similar San community who have been settled for the past 15 yr were the subjects of the present study. Anemia, due in the main to iron and/or folate deficiency, has become more common. Alcoholism has become rife in both sexes and all age groups. Our findings show that a settled lifestyle has resulted in a significant deterioration in the San's hematological nutrition.

Sera from 242 South African blacks with hepatocellular carcinoma were assayed for unsaturated vitamin B12 binding capacity (UBBC) and vitamin B12 activity. Six patients were younger than 20 years of age, and 24% were younger than 30 years of age. Eighty-four percent of the patients had a slightly raised UBBC and 86% had a slightly elevated vitamin B12 value, but in no patient was an exceptionally high UBBC present. Serum UBBC and vitamin B12 were not higher in younger patients, and raised UBBC values were not related to serum alpha-fetoprotein values. Serum UBBC and vitamin B12 concentrations were not significantly different in patients with and without coexisting cirrhosis. In none of the patients with a UBBC above 3000 pg/ml was the fibrolamellar variant of hepatocellular carcinoma present. The authors conclude that South African blacks with hepatocellular carcinoma do not secrete an abnormal vitamin B12 binding protein.

Biliary excretion of cobalamin was studied in baboons after intravenous injection of 57 Co-radio-labeled cyanocobalamin. Radioactivity appeared in bile after 20 min, and up to 4.4% of the dose was recovered in 6 h. It was estimated that 4 μg cobalamin traverses the baboon biliary tract each day. These studies in the nonhuman primate confirm that there is considerable biliary excretion of cobalamin and that enterohepatic circulation of the vitamin is important for maintenance of normal cobalamin balance.

Specific folate binding protein (FBP) and non-specific binding protein have been prepared from pooled serum from patients in late pregnancy. The two classes of folate binder were labelled with 75Se-selenofolate and injected into non-pregnant and pregnant rabbits. Selenofolate bound to specific FBP was delivered predominantly to the liver, in both pregnant and non-pregnant animals. Fetal tissue took up selenofolate from non-specific FBP only. Specific FBP thus functions to deliver folate to stores and represents a storage pool of folate. The non-specific FBP are responsible for the delivery of folate to the fetus, and probably also to sites of utilization.

Maise meal fortified with folic acid was administered to subjects in late pregnancy in a daily dose of either 500 or 300 mug of folic acid. Changes in hematological and folate nutritonal status were compared to those in subjects receiving 300 mug folic acid/day in tablet form, and also in subjects in a previous study who received unfortified maize meal (control group) or meal containing 1,000 mug folic acid/day. In all groups receiving folic acid, red cell and serum folate levels rose progressively, and the rate of rise increased with increasing doses of folic acid. Maize meal containing a daily dose of 500 mug folic acid produced an effect similar to that of 300 mug daily in tablet form. Maize containing 300 mug added folic acid daily was effective in preventing the progression of folate depletion in late pregnancy.

Fruit bats are an animal model for the neurologic damage which occurs in vitamin B12-deficient humans. Cobalamin (vitamin B12) analogs were not detected in the plasma of fruit bats treated with nitrous oxide (N2O), which inactivates cobalamin. This observation does not lend support to the suggestion that the neurological changes associated with cobalamin inactivation by N2O and/or cobalamin deficiency per se may be related to the accumulation of cobalamin analogs. However, although the plasma of control fruit bats lacked analogs, we did find analogs in their livers, at levels about 10% of total liver corrinoids, similar to human liver analog levels.

The fruit bat provides a unique small mammal model of the neurological changes associated with cobalamin deficiency. Work with this model has shown that methionine moderates the development of the neurological impairment. This action does not appear to be via the methyl donor S-adenosylmethionine, but its role in the provision of formate is not excluded. Furthermore, methylation reactions in the nervous system are not impaired in severe cobalamin deficiency, despite low levels of methionine synthetase activity. The accumulation of physiologically inactive analogues of cobalamin also do not appear to be aetiologically important in the neuropathy. Brain folates are minimally affected by severe cobalamin deficiency, although liver folates decrease significantly. Deranged GABA function in the brain may play a role in the symptomatology of cobalamin deficiency. There is some evidence for the hypothesis that deranged fatty acid metabolism in neural tissue contributes to altered membrane structure and hence function. Changes in the properties of membrane proteins may play a contributory role. The biochemical basis of the neuropathy has still to be fully elucidated.

1. Long-term exposure of the fruit bat Rousettus aegyptiacus to nitrous oxide, which inactivates methylco balamin, leads to neurological impairment and ataxia.2. In N2, O-exposed animals, liver concentrations of total folates and methyl folates decreased to less than one fifth that of control animals. Pediococcus cerevisiue-active folates were also reduced.3. In brain, there were no changes in total or methyl folates, but P.cerevisiae-active folates were lower in N2, O-exposed animals.4. Supplementation with methionine retarded the development of neurological impairment and the fall in liver total and methyl folates, but not that in P. cerevisiae-active folates.5. Supplementation with serine failed to retard the development of neurological impairment or fall in hepatic folates.6. The present results suggest that the N2O-induced neurological impairment in the bat is not related to depletion of cerebral folates, but do not exclude changes in the subcellular distribution of folates.

Summary A three-phase purification process for the separation of normal bone marrow plasma cells is described. Material was obtained from haematologically normal humans and baboons. Known monoclonal and polyclonal B cell activators were avoided both in vivo and in vitro. In Phase I, bone marrow fragments were separated from cell suspensions by buoyant density methods. Marrow fragments were shown to be richer in plasma cells than the corresponding marrow cell suspensions. Phase II consisted of culturing fragments by a simple suspension method in which a selective affinity of plasma cells and marrow stromal cells resulted in further concentration of plasma cells with discharge of haemopoietic elements. Maximum concentration of plasma cells occurred within 7 d. In phase III, fragments were disaggregated with trypsin, and the marrow stromal cells were removed by their adherence properties. The resulting non-adherent fraction comprised approximately 85% plasma cells. The process allows for direct quantitative evaluation of normal plasma cell physiology in vitro.

A method is described for the measurement of vitamin B-12 (B-12) in solid tissues by radioisotope dilution (RID) assay. The method is a modification using double extraction of a serum B-12 RID assay which uses chicken serum as the B-12 binder. The method was developed and tested using human and bat liver specimens. Double extraction was shown to be more efficient than single extraction, and resulted in complete release of endogenous liver [ 57 Co]B-12 administered to bats. Results using the RID assay in 16 human and 17 bat liver specimens were compared with those obtained using the Lactobacillus leichmannii microbiological assay. Correlation was good, but the RID assay gave higher results. Lower results using the microbiological assay appear to be due to inadequate extraction of B-12 from tissues.

Nitrous oxide, which inactivates cobalamin when administered to fruit bats, results in severe neurological impairment leading to ataxia, paralysis and death. This occurs after about 6 weeks in animals depleted of cobalamin by dietary restriction, and after about 10 weeks in cobalamin replete bats. Supplementation of the diet with pteroylglutamic acid caused acceleration of the neurological impairment - the first unequivocal demonstration of aggravation of the neurological lesion in cobalamin deficiency by pteroylglutamic acid. The administration of formyltetrahydropteroylglutamic acid produced similar aggravation of the neurological lesion. Supplementation of the diet with methionine protected the bats from neurological impairment, but failed to prevent death. Methionine supplementation protected against the exacerbating effect of folate, preventing the development of neurological changes. These findings lend support to the hypothesis that the neurological lesion in cobalamin deficiency may be related to a deficiency in the methyl donor S-adenosylmethionine which follows diminished synthesis of methionine.

The serum vitamin B12 level falls progressively during pregnancy, when measured either by microbiologic assay or by a radioisotope dilution technique. The concentration of the vitamin assayed by radioisotope dilution was consistently higher than the value obtained by microbiologic assay. This discrepancy was greatest in early pregnancy and decreased as the duration of pregnancy increased. These results add further evidence to the hypothesis that changes in serum binders for vitamin B12 are of major significance in the pathogenesis of the observed fall in serum levels of the vitamin during pregnancy.

[2-14C]Tetrahydropteroylglutamic acid and 5-[G-3H]methyltetrahydropteroylglutamic acid were given intraperitoneally to fruit bats. Folate polyglutamates were formed in the liver from both substrates in different amounts and at different rates. The methylfolate pool appeared to remain separate from the tetrahydrofolate pool. More polyglutamate was formed from tetrahydropteroylglutamic acid than from 5-methyltetrahydropteroylglutamic acid. There was a fall in the folate content of the liver in the vitamin B-12-deficient bat and a more rapid incorporation of folates into polyglutamates but thereafter a more rapid loss of the labelled folate from liver.

1. Vitamin B12metabolism was studied in bats of the speciesRousettus aegyptiacus, which live on an all-fruit diet in the wild.2. There was a wide range in values for the serum vitamin B12concentration of newly captured bats, but bats captured in the early spring had significantly higher mean serum vitamin B12levels than bats captured in the early autumn.3. There was an exponential decrease in serum vitamin B12concentration with time in captivity for bats fed on a vitamin B12-deficient, all-fruit diet; the biological half-life was 80 d in serum, 109 d in liver and 164 d in kidney.4. The main storage organ for vitamin B12in the bat was the liver, mean content 1067 ng vitamin B12. After 50 d, injected [57Co]cyanocobalamin had equilibrated with body vitamin B12stores, and 17% of the retained radioactivity was present in the liver. From these results it was calculated that the total body vitamin B12content of the bat wasc.6500 ng.5. The biological half-life of injected [57Co]cyanocobalamin was 70–88 d and the calculated daily requirement was 50–60 ng (0.2 μg/kg body-weight per d).6. As serum vitamin B12levels decreased, serum folate levels increased. The erythrocyte folate concentration increased significantly after 130 d on the all-fruit diet and then decreased to the initial values after 190 d.7. Vitamin B12metabolism in the fruit bat is similar in many respects to that of man, but on a ‘weight-for-weight’ basis the bat has a 5- to 15-fold greater requirement for this vitamin.8. Vitamin B12deficiency may be induced fairly rapidly in fruit bats fed on an all-fruit diet.

1. The effect of methylcobalamin inactivation by the anaesthetic gas nitrous oxide on the activity of the cobalamin-dependent methionine synthetase (5-methyltetrahydrofolate homocysteine methyltransferase; EC 2.1. I. 13) reaction, and on DNA synthesis, in the fruit bat Rousettus aegyptiucus, was examined.2. Methionine synthetase activity in the liver of bats exposed to N2O—oxygen (50: 50, v/v) for 90 min/d averaged 32% of that of controls after 4 d of exposure and only 5% after 12–14 weeks of exposure.3. DNA synthesis in the bone marrow, as reflected by the deoxyuridine suppression test, was unaffected by 4 d of exposure to N2O and only minimally affected after 5–10 weeks of exposure.4. These results suggest that DNA synthesis in the fruit bat is unusually resistant to inhibition of methionine synthetase and imply the existence of a non-methylated circulating folate pool in this species.

Folate in the brain of the South African fruit bat consists of 10-formyltetrahydropteroylglutamic acid and the tri-, tetra- and penta- forms of 5-methyltetrahydropteroylglutamic acid. Following parenteral injection, only 5-[3H]-methyltetrahydropteroylglutamic acid was taken up by the brain, but none of a dose of 14C-labelled tetrahydropteroylglutamic acid was detectable. Only trace amounts of the 5-methyltetrahydropteroylglutamic acid were converted into the formyl compounds and a small amount of methyltetrahydropteroyltriglutamic acid appeared after 96 h. There was no significant difference in vitamin B-12-deficient animals.

Methods for the rapid quantitation of unsaturated transcobalamin II (TC II) and R-binders in serum following separation of these binders using (a) adsorption by uncoated charcoal, (b) adsorption by Quso G32, (c) precipitation with ammonium sulphate, and (d) acidification of serum, have been compared with the standard gel filtration technique. In serum from healthy subjects and patients with elevated R-binders, the serum acidification and Quso methods yielded results in closest agreement with those obtained by gel filtration. When TC II was markedly elevated, the recommended concentration of Quso failed to remove all TC II from serum. Both the serum acidification and Quso methods are suitable for routine use, providing that with the latter method the concentration of Quso is increased when elevated levels of TC II are anticipated, as, eg, in severe jaundice. All the rapid methods tested yielded clinically significant results in patients with myeloproliferative disorders.

Summary. Transcobalamins I, II and III (TCI, TCII, TCIII) were purified from human serum, saturated with 57Co-vitamin B12 (57Co-B12), and injected into pregnant rabbits. Whole body retention of the 57Co-B12 averaged 91% and was similar for each of the three vitamin B12 binders. A maximum of 62% of the injected 57Co-B12 was found in fetal tissues when the vitamin was injected bound to TCII, but only 6–7% when bound to TCI or TCIII. Thus TCII is responsible for the delivery of vitamin B12 to the fetus.