The distribution of cobalamin cofactors was investigated in the livers and tumors of rats bearing... more The distribution of cobalamin cofactors was investigated in the livers and tumors of rats bearing transplanted Morris 7777 or 7800 hepatomas, in the livers of rats treated with the hepatocarcinogen diethylnitrosamine, and in normal rats. There was a significant increase in the proportion of methylcobalamin both in livers and tumors from rat bearing the hepatomas 7777 and 7800 compared to the proportion of methylcobalamin in the livers of normal rats. The total cobalamin content of the hepatomas was significantly lower than that of host or control livers. Similarly, the total cobalamin content of the livers from the tumor-bearing rats was less than that in control animals. The administration to rats of an acute dose of diethylnitrosamine led to an 84% increase in the hepatic concentration of methylcobalamin. Chronic administration of diethylnitrosamine slightly increased the hepatic methylcobalamin concentration, but this was not statistically significant. Liver weight was reduced, a...
Interrelationships between the B-vitamins in B12-deficiency neuromyelopathy. A possible malabsorption-malnutrition syndrome
The American Journal of Clinical Nutrition, 1975
Five patients presenting clinically with a form B12-deficiency neuromyelopathy, with cord involve... more Five patients presenting clinically with a form B12-deficiency neuromyelopathy, with cord involvement in all and proximal muscle weakness in two of them, were investigated for their neurologic, hematologic and vitamin status. Megaloblastosis and achlorhydria were present in all, and impaired absorption of 57Co vitamin B12 and of D-xylose was detected in four. Total cyanide extracted vitamin B12 (A) was lowered in all cases and noncyanide extractable (B) in four of the five, being zero in three. All five responded to injections of hydroxocobalamin. In two patients sequential estimations showed that both A and B, especially the latter, rose steeply initially, normalizing at 50% of A after some weeks. Moiety B is suggested to be physiologically the more active and dissociable form of vitamin B12. Markedly elevated initial serum folate levels, and their subsequent fall under treatment with B12, indicated the operation of the "methyltetrahydrofolate trap". Blood levels of thiamin, nicotinic acid and pantothenic acid were within normal limits. However, serum riboflavin (B2) total vitamin B6 and pyridoxal were reduced in all where tested. Vitamin B6 deficiency could have resulted from its own malabsorption and have contributed to be B12 deficiency. Vitamin B2 and B6 levels also corrected themselves on B12 therapy. The B-vitamin deficiencies in our patients probably resulted from intestinal malabsorption, with a possible factor of malnutrition consequent to their strictly vegetarian diet.
I. The B-vitamin status of fifty-nine patients, mainly from the lower socioeconomic classes in Bo... more I. The B-vitamin status of fifty-nine patients, mainly from the lower socioeconomic classes in Bombay, with a history of chronic malnutrition, and of alcoholism of 1.5-zo years' duration, was studied before and during treatment, and in relation to their clinical, especially neurological, condition. These patients were divided into two neurological categories : (I) those with peripheral neuropathy (mainly sensory and distal) alone, (2) those with mental changes (mainly confusion and disorientation) also. Both categories frequently showed pellagrous pigmentation and mucocutaneous signs of B-vitamin deficiency. 2. Thiamin and erythrocyte transketolase (EC 2. 2. I. I) activity, riboflavin, nicotinic acid, pantothenic acid, total and pyridoxal fraction of vitamin Be, folate and total vitamin BIZ were estimated in the blood and the cerebrospinal fluid (CSF) of these patients, and also in the blood of sixty-nine control subjects and in the CSF of some of them. The concentrations of all the vitamins, except vitamin B,,, were highly significantly lower in the blood of patients of category I compared to the controls, and erythrocyte transketolase activity and pyridoxal concentration in patients of category 2 were significantly lower than those of category I patients. Blood pantothenic acid and folate concentrations were reduced less consistently. 3. Serum vitamin Bla concentration was significantly increased (though within normal range) in the patients compared to the control group, probably because of the moderate hepatic insufficiency (as assessed by liver function tests) in the former. 4. The concentrations of thiamin, riboflavin, nicotinic acid and total vitamin B, were also highly significantly lower in the CSF in patients of category I compared with controls. Furthermore, thiamin, nicotinic acid and total vitamin Be concentrations were significantly lower in patients of category 2 than those of category I patients, indicating that C S F levels reflect better the neurological status of these patients. 5. There was a moderate increase in the blood concentration of all the vitamins tested, after a relatively poor hospital diet alone. There was a concurrent increase in the blood levels of thiamin, riboflavin, nicotinic acid and pantothenic acid after parenteral treatment with either thiamin or nicotinic acid. The administration of pyridoxine resulted in a significant increase in the blood levels of riboflavin and the pyridoxal fraction of vitamin B,. There is considerable information in the literature on the concentrations of various nutrients, including B-vitamins, in malnourished alcoholic patients (Fennelly, Frank, # The material included here was presented as a talk at the session on Human Nutrition, at the XXVIth International Congress of Physiology, New Delhi, October 1974. I44 D. K. DASTUR AND OTHERS I976 Baker & Leevy, 1964; Baker & Frank, 1968a), and on their neuropathological status (Victor, 1965). However, there is very little information on the levels in the cerebrospinal fluid (CSF), their correlation with those in the blood and with the neurological and mental status of the patients, and the intervitamin relationships, except in Wernicke's encephalopathy (Thompson, Frank, Baker & Leevy, 1971). Sobotka, Baker & Frank (1960) and Baker & Frank (1968b) estimated concentrations of some B-vitamins in the CSF of normal subjects. I n alcoholism there is increased vitamin requirement to compensate for decreased hepatic storage and for cellular repair (Leevy, Thompson & Baker, 1970). In a small group of patients with alcoholism, Shaw (1971) did not find any significant reduction in the folate level of the CSF Recently Pauling (1968) suggested that 'the cerebrospinal concentration of a vital substance may be grossly low' while that in the blood remains within normal limits, referring particularly to thiamin and nicotinic acid. I n view of the above considerations, as a part of our current multidisciplinary study of various nutritional disorders of the nervous system (Dastur, Wadia, Bharucha, Desai, Santhadevi, Quadros, Gagrat, Razzak, Avari & Irani, 1972), including malnutrition associated with alcoholism, simultaneous estimations of thiamin, riboflavin nicotinic acid, pantothenic acid, total vitamin B,, folate and total vitamin B,, were done in the blood and the CSF; values obtained were correlated, in the present report, by sequential estimations, with the clinical status of these patients and with the effect of treatment with different B-vitamins by reference to the results from typical patients. The metabolic interrelationships between the B-vitamins are being stressed in the present report, with special reference to the peripheral neuropathy and mental changes. The relationship between the blood and CSF levels of some of these vitamins will also be discussed.
Only 20 patients with optic neuritis of unknown aetiology could be collected in Bombay over a per... more Only 20 patients with optic neuritis of unknown aetiology could be collected in Bombay over a period of two and a half years. Only six of them were smokers and only three (one a non-smoker) had bilateral centrocaecal scotomata similar to those found In tobacco amblyopia. In 11 untreated patients the serum vitamin B1, and plasma thiocyanate levels were estimated and found to show no significant differences from those in normal control subjects. Treatment with hydroxocobalamin in the majority of cases and with cyanocobalamin, corticotrophin, or prednisolone in the rest showed equally good results; spontaneous improvement was seen in one case. No significant role could be assigned to smoking (cyanide) or to vegetarianism in the production of optic neuritis in these patients; nor was there any evidence of depletion oftotal (cyanide-extracted) B1, or of an increase in the proportion of non-cyanide-extracted B1, in the serum.
In a rat model, following exposure to rat folate receptor alpha antibodies (FRαAb) during gestati... more In a rat model, following exposure to rat folate receptor alpha antibodies (FRαAb) during gestation, FRαAb accumulates in the placenta and the fetus and blocks folate transport to the fetal brain and produces behavioral deficits in the offspring. These deficits could be prevented with folinic acid. Therefore, we sought to evaluate folate transport to the brain in young rat pups and determine what effect FRαAb has on this process, to better understand the folate receptor autoimmune disorder associated with cerebral folate deficiency (CFD) in autism spectrum disorders (ASD). When injected intraperitoneally (IP), FRαAb localizes to the choroid plexus and blood vessels including the capillaries throughout the brain parenchyma. Biotin-tagged folic acid shows distribution in the white matter tracts in the cerebrum and cerebellum. Since these antibodies can block folate transport to the brain, we orally administered various folate forms to identify the form that is better-absorbed and tran...
Food fortification and folic acid supplementation during pregnancy have been implemented as strat... more Food fortification and folic acid supplementation during pregnancy have been implemented as strategies to prevent fetal malformations during pregnancy. However, with the emergence of conditions where folate metabolism and transport are disrupted, such as folate receptor alpha autoantibody (FRαAb)-induced folate deficiency, it is critical to find a folate form that is effective and safe for pharmacologic dosing for prolonged periods. Therefore, in this study, we explored the absorption and tissue distribution of folic acid (PGA), 5-methyl-tetrahydrofolate (MTHF), l-folinic acid (levofolinate), and d,l-folinic acid (Leucovorin) in adult rats. During absorption, all forms are converted to MTHF while some unconverted folate form is transported into the blood, especially PGA. The study confirms the rapid distribution of absorbed folate to the placenta and fetus. FRαAb administered, also accumulates rapidly in the placenta and blocks folate transport to the fetus and high folate concentra...
Background: Only cobalamin carried by transcobalamin (holo-transcobalamin) is available for cellu... more Background: Only cobalamin carried by transcobalamin (holo-transcobalamin) is available for cellular uptake and hence is physiologically relevant. However, no reliable or accurate methods for quantifying holo-transcobalamin are available. We report a novel holo-transcobalamin assay based on solid-phase capture of transcobalamin. Methods: A monoclonal antibody specific for human transcobalamin with an affinity constant >1010 L/mol was immobilized on magnetic microspheres to capture and concentrate transcobalamin. The cobalamin bound to transcobalamin was then released and assayed by a competitive binding radioassay. The quantification of holo-transcobalamin was accomplished using calibrators composed of recombinant, human holo-transcobalamin. Results: The assay was specific for holo-transcobalamin and had a detection limit of 5 pmol/L. Within-run and total imprecision (CV) was 5% and 8–9%, respectively. The working range (CV <20%) was 5–370 pmol/L. Dilutions of serum were linea...
Transcobalamin II (TCII) is a plasma protein that binds vitamin B12 (cobalamin; Cbl) and facilita... more Transcobalamin II (TCII) is a plasma protein that binds vitamin B12 (cobalamin; Cbl) and facilitates the cellular uptake of the vitamin by receptor-mediated endocytosis. In genetic disorders that are characterized by congenital deficiency of TCII, intracellular Cbl deficiency occurs, resulting in an early onset of megaloblastic anemia that is sometimes accompanied by a neurologic disorder. To define the genetic basis for TCII deficiency, we have cloned and characterized the human gene that encodes this protein. The gene spans a minimum of 18 kbp and contains nine exons and eight introns, with a polyadenylation signal sequence located 509 bp downstream from the termination codon and a transcription initiation site beginning 158 bp upstream from the ATG translation start site. The 5′ flanking DNA does not have a TATA or CCAAT regulatory element, but a 34-nucleotide stretch beginning just upstream of the CAP site contains four tandemly organized 5′-CCCC-3′ tetramers. This sequence is a...
The uptake of 57Co-cyanocobalamin (CN-Cbl) and its conversion to 5- deoxyadenosylcobalamin (Ado-C... more The uptake of 57Co-cyanocobalamin (CN-Cbl) and its conversion to 5- deoxyadenosylcobalamin (Ado-Cbl), methylcobalamin (Me-Cbl), and hydroxocobalamin (OH-Cbl) has been studied in phytohemagglutinin (PHA)- transformed lymphocytes from normal subjects and patients with patients with pernicious anemia. Uptake and conversion were much greater by PHA- stimulated lymphocytes than by mature non-transformed lymphocytes. In normal cells, uptake of 57Co-CN-Cbl and synthesis of the cobalamin coenzymes were approximately linear between 3 and 48 hr incubation. Ado- Cbl was the major cobalamin formed, and after 72 hr the cells contained about twice as much Ado-Cbl as Me-Cbl. Uptake by lymphocytes from patients with untreated pernicious anemia (PA) was greater than that by normal lymphocytes, but the proportions of Ado-Cbl and Me-Cbl synthesized by each were similar. Folic acid and methyltetrahydrofolate enhanced synthesis of Me-Cbl both in normal and in PA cells, while methotrexate and 5-fluoroura...
The molecular mechanisms that underlie the neurological manifestations of patients with inherited... more The molecular mechanisms that underlie the neurological manifestations of patients with inherited diseases of vitamin B12 (cobalamin) metabolism remain to date obscure. We observed transcriptomic changes of genes involved in RNA metabolism and endoplasmic reticulum stress in a neuronal cell model with impaired cobalamin metabolism. These changes were related to the subcellular mislocalization of several RNA binding proteins, including the ELAVL1/HuR protein implicated in neuronal stress, in this cell model and in patient fibroblasts with inborn errors of cobalamin metabolism and Cd320 knockout mice. The decreased interaction of ELAVL1/HuR with the CRM1/exportin protein of the nuclear pore complex and its subsequent mislocalization resulted from hypomethylation at R-217 produced by decreased S-adenosylmethionine and protein methyl transferase CARM1 and dephosphorylation at S221 by increased protein phosphatase PP2A. The mislocalization of ELAVL1/HuR triggered the decreased expression...
Endothelial cells from human umbilical vein secrete functional transcobalamin II
American Journal of Physiology-Cell Physiology, 1989
Transcobalamin II (TCII) is a cobalamin (Cbl) binding protein in the plasma that mediates the cel... more Transcobalamin II (TCII) is a cobalamin (Cbl) binding protein in the plasma that mediates the cellular uptake of Cbl. Although the synthesis of TCII by a variety of cultured mammalian cells and by some isolated perfused organs has been reported, no single tissue has been identified as the source of TCII in vivo. In this study, we demonstrate that cultured human umbilical vein endothelial cells secrete a protein that binds CN[57Co]Cbl, elutes from a Sephacryl S-200 column in the same position as TCII, and precipitates with an antiserum to purified human TCII. The biosynthesis of TCII by these cells was confirmed by demonstrating the incorporation of [35S]methionine into a nascent protein that immunoprecipitated with anti-TCII and which, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) had an Mr of 43,000, the same as human TCII. This secreted protein also had the functional properties of TCII because it facilitated the uptake of CN[57Co]Cbl by the same endothel...
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, Jan 9, 2018
Cobalamin [Cbl (or B)] deficiency causes megaloblastic anemia and a variety of neuropathies. Howe... more Cobalamin [Cbl (or B)] deficiency causes megaloblastic anemia and a variety of neuropathies. However, homeostatic mechanisms of cyanocobalamin (CNCbl) and other Cbls by vascular endothelial cells are poorly understood. Herein, we describe our investigation into whether cultured bovine aortic endothelial cells (BAECs) perform transcytosis of B, namely, the complex formed between serum transcobalamin and B, designated as holo-transcobalamin (holo-TC). We show that cultured BAECs endocytose [Co]-CNCbl-TC (source material) via the CD320 receptor. The bound Cbl is transported across the cell both via exocytosis in its free form, [Co]-CNCbl, and via transcytosis as [Co]-CNCbl-TC. Transcellular mobilization of Cbl occurred in a bidirectional manner. A portion of the endocytosed [Co]-CNCbl was enzymatically processed by methylmalonic aciduria combined with homocystinuria type C (cblC) with subsequent formation of hydroxocobalamin, methylcobalamin, and adenosylcobalamin, which were also tran...
Autism spectrum disorder (ASD) has been linked to abnormalities in folate metabolism. Polymorphis... more Autism spectrum disorder (ASD) has been linked to abnormalities in folate metabolism. Polymorphisms in folate genes may act in complex polygenic ways to increase the risk of developing ASD. Autoantibodies that block folate transport into the brain have been associated with ASD and children with ASD and these autoantibodies respond to high doses of a reduced form of folate known as folinic acid (leucovorin calcium). Some of the same abnormalities are also found in mothers of children with ASD and supplementing folate during preconception and gestational periods reduces the risk to the offspring from developing ASD. These data suggest that folate pathway abnormalities may be a major metabolic disturbance underlying ASD that can be leveraged as biomarkers to improve symptoms and prevent ASD.
Vitamin B12 (cobalamin) deficiency is prevalent worldwide and causes megaloblastic anemia and neu... more Vitamin B12 (cobalamin) deficiency is prevalent worldwide and causes megaloblastic anemia and neurologic deficits. While the anemia can be treated, the neurologic deficits can become refractive to treatment as the disease progresses. Therefore, timely intervention is critical for a favorable outcome. Moreover, the metabolic basis for the neuro-pathologic changes and the role of cobalamin deficiency in the pathology still remains unexplained. Using a transcobalamin receptor / CD320 knockout mouse that lacks the receptor for cellular uptake of transcobalamin bound cobalamin, we aimed to determine whether cobalamin deficiency in the central nervous system produced functional neurologic deficits in the mouse that would parallel those observed in humans. Our behavioral analyses indicate elevated anxiety and deficits in learning, memory and set-shifting of a spatial memory task in the KO mouse. Consistent with the behavioral deficits, the knockout mouse shows impaired expression of the ea...
Folate receptor α (FRα) autoantibodies (FRAAs) are prevalent in Autism Spectrum Disorder (ASD). F... more Folate receptor α (FRα) autoantibodies (FRAAs) are prevalent in Autism Spectrum Disorder (ASD). FRAAs disrupt folate transport across the blood-brain barrier by binding to the FRα. Thyroid dysfunction is frequently found in children with ASD. We measured blocking and binding FRAAs and thyroid stimulating hormone (TSH), free T4 (FT4), total T3 (TT3), reverse T3 (rT3), thyroid releasing hormone (TRH) and other metabolites in 87 children with ASD, 84 of whom also underwent behavior and cognition testing and in 42 of whom FRAAs, TSH and FT4 were measured at two time points. To better understand the significance of the FRα in relation to thyroid development, we examined FRα expression on prenatal and postnatal thyroid. TSH, TT3 and rT3 were above the normal range in 7%, 33% and 51% of the participants and TRH was below the normal range in 13% of the participants. FT4 was rarely outside the normal range. TSH concentration was positively and the FT4/TSH, TT3/TSH and rT3/TSH ratios were inv...
Uploads
Papers by Edward Quadros