Uptake of cobalamin and markers of cobalamin status: a longitudinal study of healthy pregnant women.

BACKGROUND: Currently, it is unknown whether the decline in plasma cobalamin observed during pregnancy is caused by malabsorption of the vitamin. This study examined cobalamin absorption and markers of cobalamin status during normal pregnancy. METHODS: Twenty-seven pregnant Danish women were examined at gestation weeks 13, 24 and 36. The absorption test CobaSorb was performed in all women implying measurement of holotranscobalamin or cyanocobalamin bound to transcobalamin before and after 2 days intake of 3 × 9 µg cobalamin. Serum cobalamin and the two cobalamin binding proteins transcobalamin and haptocorrin, including haptocorrin saturated with cobalamin or analogues, were measured, and so was plasma methylmalonic acid and homocysteine. RESULTS: No change in the uptake of cobalamin was observed throughout pregnancy. Serum cobalamin displayed a gradual decline during pregnancy (p<0.0001), while holotranscobalamin remained unchanged, despite an increase in total transcobalamin (p<0.0001). In accord with these results, total haptocorrin showed a decline from the 1st to 3rd trimester (p=0.007) and cobalamin bound to haptocorrin declined (p<0.0001). Interestingly, the amount of cobalamin analogues attached to haptocorrin remained unchanged. Methylmalonic acid (p=0.002) and homocysteine (p<0.0001) increased during pregnancy. CONCLUSIONS: Cobalamin absorption remains unchanged during normal pregnancy, as judged by the CobaSorb test. No change was observed in the biological active holotranscobalamin during pregnancy. Thus, the pregnancy-related decline in cobalamin is caused by alternations in haptocorrin-bound cobalamin. Surprisingly, no pregnancy-related change was observed in the amount of analogues attached to haptocorrin.

The vitamin B12 absorption test, CobaSorb, identifies patients not requiring vitamin B12 injection therapy.

BACKGROUND: Treatment with vitamin B12 has virtually no side effects; however, life-long treatment is inconvenient for the patient and constitutes a cost for society. OBJECTIVE: To investigate whether vitamin B12 injection treatment reflects the actual need for treatment or whether some patients are treated unnecessarily with vitamin B12 injections. MATERIAL AND METHODS: A prospective intervention study was conducted among nine general practitioners in Western Sealand County, Denmark. Forty-four patients older than 18 years who had received injection therapy with vitamin B12 for a median of eight years (range 1-26 years) were included. After discontinuation of vitamin B12 injections, blood samples were analysed monthly for hemoglobin, cobalamin, holotranscobalamin, homocysteine and methylmalonic acid. The capacity to absorb vitamin B12 was examined after a median of 13 months (range 5-32 months) by measurement of holotranscobalamin or cyanocobalamin on transcobalamin before and after 1 and 2 days intake of 3 × 9 µg of vitamin B12. Patients unable to absorb the vitamin continued treatment with vitamin B12 injection. The remaining patients participated in a follow-up study receiving 9 µg oral vitamin B12 daily or no vitamin B12 substitution. RESULTS: Of the 44 patients studied, 35 patients were able to absorb vitamin B12. None of the patients included in the follow-up study showed biochemical signs of vitamin B12 deficiency by the end of the study. CONCLUSION: Our results suggest that the capacity for absorbing vitamin B12 should be examined prior to the choice of treatment.

Mouse transcobalamin has features resembling both human transcobalamin and haptocorrin.

In humans, the cobalamin (Cbl) -binding protein transcobalamin (TC) transports Cbl from the intestine and into all the cells of the body, whereas the glycoprotein haptocorrin (HC), which is present in both blood and exocrine secretions, is able to bind also corrinoids other than Cbl. The aim of this study is to explore the expression of the Cbl-binding protein HC as well as TC in mice. BLAST analysis showed no homologous gene coding for HC in mice. Submaxillary glands and serum displayed one protein capable of binding Cbl. This Cbl-binding protein was purified from 300 submaxillary glands by affinity chromatography. Subsequent sequencing identified the protein as TC. Further characterization in terms of glycosylation status and binding specificity to the Cbl-analogue cobinamide revealed that mouse TC does not bind Concanavalin A sepharose (like human TC), but is capable of binding cobinamide (like human HC). Antibodies raised against mouse TC identified the protein in secretory cells of the submaxillary gland and in the ducts of the mammary gland, i.e. at locations where HC is also found in humans. Analysis of the TC-mRNA level showed a high TC transcript level in these glands and also in the kidney. By precipitation to insolubilised antibodies against mouse TC, we also showed that >97% of the Cbl-binding capacity and >98% of the Cbl were precipitated in serum. This indicates that TC is the only Cbl-binding protein in the mouse circulation. Our data show that TC but not HC is present in the mouse. Mouse TC is observed in tissues where humans express TC and/or HC. Mouse TC has features in common with both human TC and HC. Our results suggest that the Cbl-binding proteins present in the circulation and exocrine glands may vary amongst species.

Holotranscobalamin remains unchanged during pregnancy. Longitudinal changes of cobalamins and their binding proteins during pregnancy and postpartum.

We confirm a decrease in cobalamins during pregnancy, and report that the active part of cobalamins (holotranscobalamin, holoTC) remains unchanged. The decrease in cobalamins is explained by a decreased holohaptocorrin (holoHC), suggesting that holoTC rather than cobalamins should be used as a marker of vitamin B12 deficiency during pregnancy.

Haptocorrin in humans.

BACKGROUND: Evolutionary haptocorrin is the youngest of the cobalamin-binding proteins. It evolved by duplication of the intrinsic factor gene and has been identified in most mammals examined. Its ability to bind both cobalamin and analogues is well established, but apart from that, our knowledge concerning its function and its distribution in adult and foetal life is limited. In this study, we present data on the tissue expression of haptocorrin and on the relation between analogues on haptocorrin and vitamin B(12) status in humans. METHODS: Polyclonal antibodies towards haptocorrin were used to study the localisation in foetal and adult tissues by immunohistochemistry. Positive immunoreactions were primarily observed in exocrine glands, the gastrointestinal tract and the respiratory system. ELISA was used for measurement of holo- and total haptocorrin in blood samples from individuals diagnosed with vitamin B(12) deficiency, based on measurement of methylmalonic acid (micromol/L) as evident (>0.75, n=61), suspected (0.29-0.75, n=155) or not present (<0.29, n=170). Cobalamins and holotranscobalamin were measured in the same individuals. RESULTS: Holohaptocorrin was considerably higher than holohaptocorrin-cobalamins (cobalamins minus holotranscobalamin). The median (25th-75th percentile, pmol/L) for holohaptocorrin analogues (holohaptocorrin minus holohaptocorrin-cobalamins) was higher in deficient [200 (130-240)] compared to the non-deficient [140 (80-200)] individuals (analysis of variance and Tukey's multiple comparison test, p<0.01). CONCLUSIONS: Our results indicate that haptocorrin is widely distributed also in foetal tissues and suggest analogues to accumulate on haptocorrin in vitamin B(12)-deficient individuals, a result that warrants further studies employing methods directly measuring cobalamins and analogues attached to haptocorrin.

Plasma holotranscobalamin compared with plasma cobalamins for assessment of vitamin B12 absorption; optimisation of a non-radioactive vitamin B12 absorption test (CobaSorb).

BACKGROUND: A recently developed non-radioactive vitamin B(12) absorption test (CobaSorb) was further explored to identify the best marker for reflection of vitamin B(12) absorption and to determine the duration of the test. METHODS: Seventy-eight healthy individuals (age 21-81 years) were given three oral doses of 9 microg vitamin B(12) per day for 5 successive days. Non-fasting blood samples were collected on days 1 to 5 before administration of vitamin B(12) and on day 8. Cobalamins and holotranscobalamin were measured. RESULTS: Performance of the vitamin B(12) absorption test was evaluated in individuals with holotranscobalamin or cobalamins below the 75% percentiles. We used a change greater than 2xCV(day-to-day) in holotranscobalamin (22%) and cobalamins (12%) to indicate a change caused by absorption of vitamin B(12). Among individuals with a baseline holotranscobalamin below the 75% percentile (<75 pmol/L, n=57), 98% had an increase in holotranscobalamin >22% from day 1 to day 3. In contrast, only 72% of the individuals with baseline cobalamins below the 75% percentile (<335 pmol/L, n=57) had an increase in cobalamins >12%. CONCLUSIONS: In healthy individuals with baseline holotranscobalamin <75 pmol/L, vitamin B(12) absorption is well reflected by an increase in holotranscobalamin after 2 days administration of oral vitamin B(12).

Effect of vitamin B12 treatment on haptocorrin.

BACKGROUND: Haptocorrin (HC) carries the major part of circulating cobalamin, but whether HC is altered on treatment with vitamin B12 remains unknown. METHODS: Our study included 3 populations: a population of vegan men (n = 174; vegan population), of whom 63 were treated daily with 5 mg of oral vitamin B12 for 3 months; a group of patients with a previous methylmalonic acid (MMA) concentration >0.4 micromol/L (n = 140; population with suspected deficiency), of which 69 were treated with weekly vitamin B12 injections (1 mg) for 4 weeks; and a subgroup of participants in a vitamin B intervention study (n = 88; nondeficient population), of whom 45 were treated daily with 0.4 mg of oral vitamin B12 for 3 months. Total HC and holoHC were measured by ELISA. Cobalamin was measured by an intrinsic factor (IF)-based assay. Samples were collected at baseline and 3 months after start of treatment. RESULTS: Compared with baseline results for the 3 study populations, total HC and holoHC increased 30 pmol/L for every 100 pmol/L increase in cobalamin. After treatment with vitamin B12, holoHC (P <0.0001) and total HC (P <0.0001) increased significantly in the vegan population. Only holoHC increased in the population with suspected deficiency (P <0.0001), whereas no alteration was observed in the nondeficient population. CONCLUSIONS: The HC concentration is decreased in severely cobalamin-deficient individuals and increases on treatment. The concentration of cobalamin also relates significantly to the HC concentration in nondeficient individuals.

The cobalamin-binding proteins transcobalamin and haptocorrin in maternal and cord blood sera at birth.

BACKGROUND: Two proteins carry vitamin B12 in plasma. Transcobalamin (TC) carries approximately 25% of total plasma vitamin B12 and is 6% to 20% saturated with cobalamin. Haptocorrin (HC) binds approximately 80% of total cobalamin and is largely saturated with cobalamin. METHODS: We investigated the distribution and the relationship between concentrations of cobalamin, total and holo forms of TC, and HC in blood samples from pregnant women just before delivery (n = 92) and in cord blood samples from their newborn babies. We also investigated the relationship between these proteins and concentrations of methylmalonic acid (MMA), the functional marker of vitamin B12 status. RESULTS: Concentrations of total serum cobalamin, total HC, holoHC, and percentage of HC saturation were higher in cord blood than in the maternal blood (mean cobalamin, 268 vs 188 pmol/L; total HC, 648 vs 538 pmol/L; holoHC, 441 vs 237 pmol/L; HC saturation, 70% vs 47%). Moreover, total TC was low in cord blood, whereas both holoTC and TC saturation were higher in cord blood than in the maternal blood (mean total TC, 654 vs 1002 pmol/L; holoTC, 118 vs 53 pmol/L; TC saturation, 19.8% vs 5.4%). Higher maternal serum cobalamin was associated with higher cord blood holoTC and TC saturation (P <0.05). Gestational age was also a significant determinant of baby total TC, TC saturation, total HC, and holoHC. CONCLUSION: The close correlation between the amounts of holoTC present in cord blood and in maternal serum supports the importance of maternal cobalamin status for ensuring a sufficient supply to the baby.

Evaluation of the technical performance of novel holotranscobalamin (holoTC) assays in a multicenter European demonstration project.

A commercially available holotranscobalamin (holo-TC) radioimmunoassay (RIA) (Axis-Shield, Dundee, Scotland) was evaluated in four laboratories and compared with a holoTC ELISA run in one laboratory. The performance of the holoTC RIA assay was comparable in three of the four participating laboratories. The results from these three laboratories, involving at least 20 initial runs of "low", "medium" and "high" serum-based controls (mean holoTC concentrations 34, 60 and 110 pmol/L, respectively) yielded an intra-laboratory imprecision of 6-10%. No systematic inter-laboratory deviations were observed on runs involving 72 patient samples (holoTC concentration range 10-160 pmol/L). A fourth laboratory demonstrated higher assay imprecision for control samples and systematic deviation of results for the patient samples. Measurement of holoTC by ELISA showed an imprecision of 4-5%, and slightly higher mean values for the controls (mean holoTC concentrations 40, 70 and 114 pmol/L, respectively). Comparable results were obtained for the patient samples. The long-term intra-laboratory imprecision was 12% for the holoTC RIA and 6% for the ELISA. In conclusion, it would be prudent to check the calibration and precision prior to starting to use these holoTC assays in research or clinical practice. The results obtained using the holoTC RIA were similar to those obtained using the holoTC ELISA assay.

Glycosylation independent measurement of the cobalamin binding protein haptocorrin.

BACKGROUND: Haptocorrin carries the major part of the circulating vitamin B12. The protein is heavily glycosylated and this may have implications for its measurement. METHODS: We used two different ELISA assays. In one assay, we employed antibodies against native HC and no pre-treatment of samples or calibrators. In the other assay, we used antibodies raised against deglycosylated HC, and deglycosylated the samples and calibrators by treatment with neuraminidase and PNGase prior to analysis. Plasma samples from healthy donors were analysed. RESULTS: The ELISA against native HC showed a high detection limit (71 pmol/l) and a poor linearity for serial dilutions of samples. The ELISA against deglycosylated HC showed a detection limit of 1.6 pmol/l, an excellent linearity between 1.6 and 100 pmol/l (r(2) = 0.99) and an inaccuracy of 5% for concentrations ranging from 250 to 840 pmol/l. The 95% reference interval was 240-680 pmol/l (n = 148). The concentration of HC showed a strong association to plasma cobalamins (p < 0.0001). CONCLUSIONS: An ELISA against native HC does not ensure an equimolar measurement of HC, while this is the case when a glycosylation independent assay is employed. Using this assay, a very strong correlation between total plasma HC and cobalamins in healthy donors is obtained.