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Biomedical subjects

J A Begley

Publications and source records attributed to J A Begley.

33 records · Page 2Linked to original sources
PubMed1981

The content, binding, and forms of vitamin B12 in milk.

The vitamin B12 (cobalamin, Cbl) content of 19 human milks ranged from 0.33 to 3.20 ng/ml, mean 0.97 ng/ml. The milk content of 10 mothers taking 5 to 100 micrograms of supplemental cyanocobalamin daily was not significantly different from that of unsupplemented mothers. The Cbl native to milk was bound to an R type binder. The R binder was also the dominant, by far, unsaturated Cbl binder, but transcobalamin II was found in every milk. The amounts of transcobalamin II were of the same order of magnitude as in serum and seemed to increase with the interval postpartum. Methylcobalamin was the most abundant Cbl of milk. Human milk from well fed mothers contains adequate amounts of Cbl, but the Cbl may become available only if there are sufficient proteolytic enzymes to release it from binding to R binder.

Chromatography, Gel↗
PubMed1981

Forms of vitamin B12 in radioisotope dilution assays.

Since the presence of analogues of vitamin B12 (B12, cobalamin, Cbl) has been postulated as the basis for the high values obtained by some radioisotope dilution assays (RIDA) of serum Cbl we examined serum for analogues. None could be demonstrated in the extracts of serum prepared for RIDA as sought by both direct and indirect techniques. The natural forms of serum Cbl were converted to cyanocobalamin (CN Cbl) by this process of extraction which included cyanide (CN). The correctly performed RIDA for Cbl based on R binder gave higher values than a RIDA based on intrinsic factor or than by bioassay. By exclusion, the difference appeared to be due to unidentified factors rather than the presence of analogues.

Chromatography, Gel↗
PubMed1979

An assay for intrinsic factor based on blocking of the R binder of gastric juice by cobinamide.

An in vitro assay for measurement of gastric juice intrinsic factor (IF) was developed based on the ability of the cobinamide (Cbi) [(CN, OH) Cbi] to bind to the gastric juice R-type binders of cobalamin (Cbl) and not to the IF binder. Subsequently added radioactive Cbl, CN-[57Co] Cbl, binds only to the IF binders and allows for direct measurement of this Cbl binding protein. This Cbi blocking assay was found to function as well as the more conventional methods of IF measurement, G-100 column chromatography, and IF blocking antibody assay. The present assay has the advantage of eliminating the need for elaborate forms of protein separation and does not rely on a source of antibody.

Binding Sites, Antibody↗
PubMed1979

The forms and transport of plasma cobalamins in normal man and in myeloproliferative states.

Seven patients with MP and four controls were injected with 0.04 to 0.05 microgram of [57Co] cyanocobalamin i.m. or i.v. in order to study the shift of binding with time and the conversion of one Cbl to another. The initial pattern of binding reflected the proportion of the apo forms of TC II and R binders. In MP there was more initial binding of the injected Cbl to R-type binders and less to TC II. During the first 48 hr after intake, the injected Cbl remained mostly as cyanocobalamin. Some adenosylcobalamin appeared transiently in both control and MP subjects. Radioactive methylcobalamin did not appear in the circulation until after 48 hr, and the conversion of cyanocobalamin to methylcobalamin within the circulation was greater in MP subjects. Serum from some subjects converted small amounts of cyanocobalamin to all other forms in vitro by a heat-stable, extracellular property which was abolished by dialysis of the serum. This property of serum could have accounted for the early conversion to adenosylcobalamin but not, at least as an in vitro phenomenon, to the late appearance of methylcobalamin. Although the expected increases and abnormal patterns of R binders were observed in MP, these abnormalities could not be correlated with the increased conversion to plasma methylcobalamin. During the first 6 hr after injection the R binder-Cbl designated as alpha 2-R-Cbl disappeared from the circulation at a rate faster than that of alpha 1-R-Cbl. Subsequently the alpha 2 and alpha 1 components of R binder-Clb cleared at the same rate, and this rate was the same for both control and MP subjects.

Carrier Proteins↗
PubMed1978

Preparation of radioactive labelled (57Co) sulfitocobalamin.

Farquharson and Adams (Br. J. Nutr. 36, 127-135 (1976)) have identified sulfitocobalamin (SO3-Cb1) as one of the naturally occurring cobalamins (Cb1s) in foods. We have devised a method of making radioactive labelled SO3-Cb1 for in vivo and in vitro studies of this form of Cb1. 57Co labelled cyanocobalamin (57Co CN-Cb1) was acid photolyzed to 57Co hydroxocobalamin (57Co OH-Cb1) followed by ligand substitution with SO3-2 ion from aqueous sodium (meta) bisulfite in the dark. The resulting 57Co SO3-Cb1 was purified by organic extraction and cation exchange chromatography. The final preparation was greater than 99% 57Co SO3-Cb1 with an overall yield of greater than 70%, stable for up to four weeks at 20 degrees C in the dark, and capable of binding to the human Cb1 binding proteins Transcobalamin II (TC II), Intrinsic factor (IF) and Salivary R. This method allows a simple 1 day preparation of high specific activity labelled 57CO SO3-Cb1 for biological studies.

Cobalt Radioisotopes↗
PubMed1977

A radioimmunoassay for the R-type binders of cobalamin.

A radioimmunoassay of R-type binders of cobalmin was devised and tested. The assay incorporated an antibody against purified human salivary R binder as the binding reagent. The labeled ligand was cyano[57Co]cobalamin bound to the R binder of pooled human saliva. The standard source of unlabeled ligand was also from human saliva of known R binder content. The assay was responsive to R binders of several sources, to either pure R or that of crude sources and equally to R binder saturated or unsaturated with cobalamin. It was not responsive to transcobalamin II. The assay was reproducible and reliable.

Antibody Specificity↗
PubMed1977

Radioimmunoassay for serum transcobalamin II.

A radioimmune assay for transcobalamin II (TC II) was devised from the following: (1) TC II-57Co B12 precipitated from normal serum with (NH4)2SO4 as the labeled ligand; (2) the TC II of whole serum as the standard source of TC II; (3) rabbit anti-pure TC II as the binding agent; (4) separation of the bound and free TC II-B12 by precipitation of the antibody bound with polyethylene glycol. The assay was responsive to either TC II or TC II-B12 and to TC II either pure or in crude preparations. It was not responsive to R-type binders of B12. The median TC II of 10 normal sera was 890 pg. per milliliter and of 10 sera from 10 random hospital patients was 1,010 pg. per milliliter. There was no measurable TC II in the serum of a child with congenital absence of TC II and the assay measured levels greater than 5,000 pg. per milliliter in abnormalities of TC II metabolism.

Antigen-Antibody Complex↗
PubMed1977

Congenital deficiency of human R-type binding proteins of cobalamin.

A family expressing the congenital absence of the R-type binders of cobalamin (Cbl), vitamin B-12, was restudied. The capacity to bind Cbl to R type binders was absent from serum, saliva, cerebrospinal fluid, gastric juice granulocytes, and granulocyte output of the propositus. Serum R did not carry Cbl in vivo. There was no immunological R binder in his saliva, but cross reacting material was detected in his serum. Evidence of a partial expression of the defect was observed in offspring of two affected persons. There were no obvious clinical consequences of the defect.

Blood Proteins↗
PubMed1975

Measurement of vitamin B12-binding proteins of plasma. II. Interpretation of patterns in disease.

The technique described in the preceding paper was applied to 12 abnormal sera selected for their increase in one or more B12-binding proteins. Even in the presence of large amounts of R-type binder, the ammonium sulfate technique gave a reliable separation of R binding proteins from TC II. Measurement of the TC II in abnormal sera gave results identical to those obtained by the more standard gel filtration. The R binders of four subjects with myeloproliferative disease were further separated into alpha2-R and alpha1-R. The pattern of B12 binding of polycythemia vera (PV) was an exaggeration of the normal pattern. Binding to alpha2-R was three to four times that to alpha1-R, although the total amounts bound to both were increased. In chronic myelogenous leukemia (CML), both alpha2-R and alpha1-R were also increased, but in contrast to binding in normal sera, alpha1-R predominated. In order to interpret the findings, either whole serum R or alpha1-R and alpha2-R from patients with myeloproliferative disease were subject to isoelectric focusing. Alpha2-R consisted pricipally of components isoelectric at pH 2.9, 3.0, and 3.1. These components were present in only minor amounts in normal serum and were somewhat increased in the serum of PV. These components were very much increased in the serum of CML and predominated. Alpha2-R consisted of those components isoelectric at pH 3.4,3.6, and 4.0. These components predominated in the unsaturated binding capacity of normal sera and that of PV. It was concluded that the division of plasma R binders into alpha1-R and alpha1-R by the technique described provided information useful in the study of myeloproliferative diseases.

Ammonium Sulfate↗
PubMed1975

Measurement of vitamin B12-binding proteins of plasma. I. Technique.

The unsaturated binding capacities (UBBC) of individual vitamin B12-binding proteins in plasma were measured by a two-step procedure. Transcobalamin II (TC II) was separated by precipitation with ammonium sulfate; the "R"-type binders remaining soluble were then divided into two components by bath separation with anion exchange on DEAE-cellulose. The two R components were designated alpha1-R (TC 1) and alpha2-R (third binder, fetal binder, PV binder, TC III). Ten normal sera were studied by this technique giving a separation into TC III and total plasma R identical to that obtained simultaneously by gel filtration. The mean UBBC of TC II was 969 plus or minus 204 pg of 57 Co B12 per ml of serum. The mean contamination of the precipitated TC III with plasma R was 3%. The UBBCs of alpha 2-R and alpha 1-R were 127 plus or minus 42 and 40 plus or minus 12 pg/ml, respectively. The mean contamination of the R fraction by TC II was 14% as evaluated by gel filtration. By isoelectric focusing it was found that the alpha1-R contained principally those components isoelectric at pH isoelectric at pH of 2.9-3.2, while alpha2-R was made up of those components isoelectric at pH of 3.6 or greater.

Ammonium Sulfate↗