Int. J. Vitam. Nutr. Res.

, 69 (6), 1999, 412418

Bioavailability of Dried Asakusanori (Porphyra tenera) as a Source of Cobalamin (Vitamin B12)

Keiko Yamada1, Yoko Yamada2, Morimichi Fukuda3 and Shoji Yamada2
of Liberal Arts and Sciences, School of Health Sciences, Sapporo Medical University, South-1 and West-17,, Sapporo 060-8556, Japan 2Department of Home Economics, Faculty of Education, Hokkaido University of Education, Ainosato, Sapporo 002-8075, Japan 3Department of Laboratory Diagnostic Ultrasound and Medical Electronics, School of Medicine, Sapporo Medical University, Sapporo 060-8556, Japan Received for publication: November 4, 1998
1Department

Abstract: We have already reported that raw nori (Porphyra tenera) contains cobalamin (Cbl) but not Cbl analogues (J. Nutr. Sci. Vitaminol., 42, 497, 1996). It seems, therefore, that it is an excellent natural vegetable source of Cbl. On the other hand, it has been reported that the Cbl nutritional status of vegetarian children deteriorated as estimated by the hematological index, mean corpuscular volume (MCV), after they had dried nori as a source of Cbl. Such a discrepancy between raw and dried nori as a source of Cbl led us to investigate whether Cbl in dried nori had different properties from that in raw nori. We found that contents of Cbl homologues determined by a bioassay method in both raw and dried nori were similar. The urinary methylmalonic acid excretion increased when human female volunteers were given 40 g of dried nori daily during the test period. On the other hand, the urinary methylmalonic acid excretion did not change when volunteers were daily given 320 g of raw nori, which was equivalent to 40 g of the dried one on the basis of dehydrated weight, during the test period. By paper chromatography, 65% of the Cbl homologues were found to be comprised of Cbl analogues in dried nori, while 73% of the Cbl homologues in the raw nori were genuine Cbl. These results were confirmed by the finding that the bioassay method gave higher values for Cbl homologues than those obtained by a competitive binding assay method using an intrinsic factor as a Cbl-binding protein. Our present data demonstrated that Cbl in raw nori can be changed into harmful Cbl analogues by the drying process. Key words: Bioavailability, dried asakusanori, cobalamin

Introduction
Cobalamin (Cbl) is synthesized exclusively by bacteria and is present in normal liver but not in plants. Therefore Cbl is called an animal protein factor. There are an increasing number of reports concerning Cbl deficiency in

infants of vegetarians [1, 2]. The Cbl status of long-term adherents of a strict, uncooked vegan diet [3], the young dieter or old person whose intake of animal food is decreased is compromised. For the purpose of finding a vegetable food containing Cbl, we determined the Cbl homologue contents microbiologically using Escherichia coli

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215 in each of the seven species of seaweeds. Some seaweeds apparently contained as much as one tenth the amount of that in the bovine liver. Most of them, however, contained Cbl analogues, not Cbl. The exception was the raw nori family, for example, asakusanori (Porphyra tenera) or maruba-amanori (Porphyra suborbiculata), which contained Cbl but not Cbl analogues [4]. It seems, therefore reasonable to consider that the nori family is a good natural source of Cbl as a vegetable food. On the other hand, Dagnelie et al [5] have reported that the hematological status of Cbl-deficient children deteriorated after they used nori as a source of Cbl. Such apparent discrepant effects of raw and dried nori on Cbl nutriture led us to investigate whether Cbl in dried nori had different properties from that in raw nori. To determine this, we carried out the following experiments: 1. we estimated the Cbl homologue contents in both raw and dried nori by a microbiological method using Escherichia coli 215 and determined methylmalonic acid excreted in urine of volunteers who were given a substantial quantity of dried nori. 2. We determined genuine Cbl and Cbl analogues separately in both raw and dried nori by paper chromatography. Cbl analogues were also estimated based on the difference between the Cbl homologue content of nori determined by bioassay methods and the Cbl content determined by a competitive binding assay method. We obtained results demonstrating that Cbl in raw nori can be changed into harmful Cbl analogues by the drying process.

study protocol was approved by a medical ethical committee. Experimental diet: Experimental diets were as follows: Exp. 1: 13 days, ordinary diet consumed routinely in Japan; 4,5 days, ordinary diet + 5 g of L-Val; 69 days, ordinary diet + 5 g of L-Val + 40 g of dried asakusanori; 1016 days, ordinary diet. Exp. 2: 12 days, ordinary diet; 36 days, ordinary diet + 5 g of L-Val + 320 g of raw asakusanori; 79 days, ordinary diet. Extraction of vitamin B12: In this report, the term vitamin B12 is used as a synonym for Cbl homologues. Vitamin B12 of raw and dried asakusanori was heat extracted in ethanol with N-ethylmaleimide and acetic acid [6]. Extracts were desalted by silanised silca gel (Merck, Germany) column chromatography before analysis. Analysis of vitamin B12: Vitamin B12 was assayed microbiologically using E. coli 215 [7]. We also used a competitive binding assay method employing a Chemilumi ACS B12 kit (Corning) for some samples [8]. Determination of methylmalonic acid in urine: Urine was collected from subjects before breakfast on the morning after they were fed the experimental diets because even ordinary everyday meals have been reported to cause an increase in urinary methylmalonic acid excretion [9]. Urine was first assayed for creatinine and methylmalonic acid in urine was expressed as mol per mmol of creatinine to avoid the variation due to fluctuation of urine volume with individuality or day [10]. Determination of methylmalonic acid was performed by the method of Gutteridge and Wright [11] with slight modifications. Methylmalonate in urine was extracted with ethyl ether and the extract was applied on a Silica gel G thin-layer plate (Merck, Germany). The developing solution was 95% EtOH:25% ammonia water:water (25:4:3). After the solvent was removed by evaporation in a stream of cold air, Fast Blue B reagent (0.5% Fast Blue B in 75% ethanol:glacial acetic acid (100:4) was sprayed on the plate. On careful drying in a stream of warm air, the methylmalonic acid appears after a few minutes as a distinct purple band. When the maximum intensity of the purple color was reached, the profile of the band was copied on a transparent sheet by a black- and white-photocopier since the color rapidly fades after a few hours. The densitometric analysis of the band was made and recorded on a chart as optical density. The area of a certain peak on the chart was calculated by multiplying the half of height of the peak by the width of its bottom. The peak area and the concentration of methylmalonic acid were found to have a close relationship, with a correlation coefficient of over 0.990.

Materials and Methods

Materials: Methylcobalamin (Me-Cbl), 5-deoxyadenosylcobalamin (Ado-Cbl), cyanocobalamin (CN-Cbl) and hydroxocobalamin (OH-Cbl) were obtained from Sigma, USA. Cobinamide was from Calbiochem, USA. L-Val was from Wako Pure Chemical Industries, Ltd, Japan. Raw asakusanori (Porphyra tenera) was purchased from a fishmonger within 48 h after harvesting. Dried asakusanori was purchased from department stores. Subjects: In experiment 1, six healthy female volunteers were studied, all were college students aged 22. In experiment 2, four healthy volunteers (two women and two men, ages 5053) were studied. In all 10 subjects, erythrocyte counts, leukocyte counts, differential leukocyte counts, platelet counts and the concentration of hemoglobin were all within normal limits, as were results of renal function and liver function tests. All were in a good nutritional state, with a normal protein intake. None took drugs. Subjects were informed of the purpose of the study. The

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Separation of Cbl and Cbl analogues and identification of Cbl forms by paper chromatography: Four forms of Cbl (CN-Cbl, OH-Cbl, Me-Cbl and Ado-Cbl) and the seaweed extract were separated by paper chromatography using a developing solution of 2-butanol-28% ammonia water-water (75:5:25). After the paper was dried thoroughly, it was cut into 35 segments, each 7 mm long. The water extract from each of the 7-mm segments was analyzed by bioautography according to the method of Gimsing and Nex [12]. All procedures were done in a dark room.

OH-Cbl, Me-Cbl and Ado-Cbl. E. coli 215 can respond not only to Cbls but also to cobamides lacking 5,6dimethylbenzimidazole and cobinamide lacking the nucleotide portion of the Cbl molecule [13] in Cbl analogues. The contents of Cbl homologues (g per 100 g of dry weight) determined by a bioassay method in both raw and dried asakusanori were similar. Water contents of both raw and dried asakusanori were obtained from Standard Tables of Food Composition in Japan and utilized to compensate the Cbl concentration on dry weight base. The variation was large among individual samples in the same seaweed species. Urinary excretion of methylmalonic acid by subjects administered raw or dried asakusanori: Next, we determined urinary excretion of methylmalonic acid to examine the Cbl nutritional status of subjects who ate dried or raw asakusanori. Branched amino acids such as valine or isoleucine are converted to methylmalonyl Co A via propionyl Co A. Succinyl Co A, which is the intermediate member of TCA cycle, is formed from methylmalonyl Co A by an intramolecular rearrangement. The rearrangement reaction is catalyzed by methylmalonyl Co A mutase with Ado-Cbl as a cofactor. In Cbl-deficient subjects, the accumulation of methylmalonyl Co A occurs. Because the compound is toxic it is soon converted into methylmalonic acid and excreted into urine. This is the reason why the increase of urinary methylmalonic acid can be an indicator of Cbl deficiency [14]. Methylmalonic acid in urine was expressed as mol per mmol of creatinine to avoid the variation due to fluctuation of urine volume with individuality or day. To detect the Cbl nutritional defect clearly, 5 g of L-valine was given orally to the subjects while collecting urine [14]. The urinary methylmalonic acid excretion increased when subjects were given 40 g of dried asakusanori daily

Results
Change of the contents of Cbl homologues of asakusanori after the drying process: The Cbl homologue content of raw and dried asakusanori (Porphyra tenera) measured by the microbiological assay method using E. coli 215 is shown in Table I. In this report, the term Cbl homologue refers to both Cbls that are active in mammals and Cbl analogues that are inactive as Cbl in mammals, but are Cbl-active in microorganisms. Cbls include CN-Cbl,
Table I: Cbl homologue content of raw and dried asakusanori (Porphyra tenera) measured by microbiological assay method using E. coli 215 Cbl content (g/100 g dry weight) Raw asakusanori (n = 6) Dried asakusanori (n = 6) *analyzed t-test 12.5 5.6 P > 0.5* 14.4 2.9

Table II: Urinary excretion of methylmalonic acid by subjects administered either dried or raw asakusanori (Porphyra tenera) Urinary methylmalonic acid excretion (mol/mmol creatinine) Experiment 1 Ordinary meal (13 days) (n = 18) Ordinary meal + Val (4,5 days) (n = 12) Val + Dried asakusanori (69 days) (n = 24) Ordinary meal (1016 days) (n = 42) Experiment 2 Ordinary meal (1,2 days) (n = 8) Val + Raw asakusanori (36 days) (n = 16) Ordinary meal (79 days) (n = 12) 0.691 0.291 0.633 0.206 1.222 0.606 0.807 0.353 0.803 0.111 0.843 0.170 0.718 0.156 P < 0.01* P < 0.05*

P > 0.5* P > 0.05*

Mean values and the standard deviations for each administration were calculated from the values obtained from all urine samples (n) collected every day during the corresponding administration period. *analyzed t-test. Int. J. Vitam. Nutr. Res., 69 (6), 1999, Hogrefe & Huber Publishers

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during the test period. On the other hand, it did not change when subjects were given 320 g of raw asakusanori (corresponding to 40 g of dried asakusanori on the basis of dehydrated weight) daily (Table II). Eating of 5 g of L-valine alone did not effect urinary methylmalonic acid excretion in the group in experiment 1. Separation and characterization by paper chromatography of Cbl and Cbl analogues: Four authentic forms of Cbl were clearly separated in the order Me-Cbl (relative mobility: Rf = 0.39), CN-Cbl (Rf = 0.27), Ado-Cbl (Rf = 0.18) and OH-Cbl (Rf = 0.10) from the front. Two spots besides these four Cbls were found, one between CN-Cbl and Me-Cbl and the other in front of Me-Cbl in extracts from both dried and raw asakusanori. The compounds corresponding to these spots were not Cbls but

were Cbl-active on E. coli 215, so we named them analogue-0.30 (Rf = 0.30) and analogue-0.45 (Rf = 0.45), respectively according to their relative mobilities. Figure 1 shows the distribution of Cbl activity of Cbl homologues on E. coli 215 in raw (n = 5) and dried (n = 5) asakusanori. CN-Cbl is not a natural form and was not found in asakusanori. Sixty-five percent of the Cbl homologues were found to be present as Cbl analogues in dried nori, while 73% in raw nori were genuine Cbl. But 23.9% was present in raw asakusanori as analogue-0.45, which was inactive in humans. This value was much larger than that in our previous report [4]. Comparison of Cbl homologue content of asakusanori determined by bioassay method with Cbl content determined by competitive binding assay method: To confirm the results obtained by paper chromatography, we analyzed the content of Cbl in both raw and dried asakusanori by bioassay and competitive binding assay methods. Apparent Cbl content of asakusanori determined microbiologically by using E. coli 215 was Cbl plus its analogues, while that determined by the competitive binding assay method using pure intrinsic factors was genuine forms of Cbl. Therefore, if the values obtained by both methods coincide, all of the Cbl homologues in the sample would be genuine Cbl. In case of the raw asakusanori, the values determined by both methods were essentially the same (Fig. 2) (Y = 0.91X + 0.11, where Y is the value obtained by the competitive binding assay method and X is that from the microbiological assay method). On the other hand, in dried asakusanori, the values for Cbl homologues obtained by the bioassay method were higher than those obtained by the competitive binding assay method (Y = 0.57X + 0.28). These results indicated that dried asakusanori contained Cbl analogues and were in agreement with the results obtained by paper chromatography. Similar results were obtained with another competitive binding Cbl assay method using an M-vitamin B12/folic acid Corning kit (Corning Co. Ltd., USA) (data not shown).

Figure 1: Distribution of cobalamin (Cbl) activity on E. coli 215 in raw and dried asakusanori (Porphyra tenera) among four forms of cobalamin and their analogues. Separation and identification of Cbls and their analogues was performed by paper chromatography as described in the text. Results are means S.E. for 5 experiments and are expressed as percentages of total Cbl homologues. Results are means S.E. for 5 experiments. OH-Cbl, hydroxocobalamin; Ado-Cbl, 5deoxyadenosylcobalamin; CN-Cbl, cyanocobalamin; Me-Cbl, methylcobalamin, Analogue-0.30 and Analogue-0.45; Rf of spot = 0.30 and 0.45, respectively.

Discussion
We have already reported that raw nori (Porphyra tenera) contains Cbl but not Cbl analogues, and therefore it is a good natural vegetable source of Cbl [4]. But there are reports that the hematological status of Cbl-deficient vegetarians deteriorated after they had nori as a source of Cbl [5] and the routine ingestion of seaweeds does not supply enough Cbl to meet the bodys need [3]. In our present report, the urinary methylmalonic acid excretion, an indi-

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Figure 2: Correlation between cobalamin (Cbl) homologue content of asakusanori (Porphyra tenera) determined by bioassay method and that determined by competitive binding assay method. Cbl (and its homologue) concentration of each asakusanori extract was determined by both bioassay method using E. coli 215 and by a competitive binding assay method, chemilumi ACS B12 kit, with pure intrinsic factor as a binding protein. Assay methods of Cbl were described in the text.

cator of Cbl deficiency, did not increase when volunteers were given raw asakusanori but did do so when they were given dried asakusanori. Although the TLC method for the determination of methylmalonic acid is less sensitive than the current HPLC or GC-MS methods, we were able to detect a significant increase of urinary methylmalonic excretion by feeding subjects dried asakusanori, but not the raw one, with the aid of an oral valine load [14]. The separation of organic acids by TLC is excellent in our experience, and so the specificity of the method is sufficient. By paper chromatographic analysis, we found that 65% of the Cbl homologues in dried asakusanori presented as Cbl analogues while 73% in raw nori was genuine Cbl. These results demonstrated that Cbl in raw nori can be changed into harmful Cbl analogues by the drying process and Cbl analogues worsen the situation of Cbl nutriture. Raum et al [3], Dagnelie et al [5] did not refer to whether they used raw nori or dried nori as a source of Cbl. However, their results can be explained if they used dried nori. In addition to the microbiological assay using E. coli 215 [7] for the analysis of Cbl, we also employed a Chemilumi ACS B12 assay method (Corning Co. Ltd., USA) and radioisotope dilution assay method (RIDA) using a Vitamin B12 Corning kit (Corning Co. Ltd., USA) for some

samples. The latter two methods utilize the competitive inhibition of binding of the added labeled Cbl to Cbl in the samples for Cbl binding protein. Herbert and Drivas [15] assumed that RIDA measured the total Cbl, i.e. Cbl plus analogues of Cbl, by using haptocorrin (also called R-binder) and Cbl by using an intrinsic factor as a ligand binding protein. The difference of total Cbl and Cbl represents analogues. Based on his assumption, he reported that more than 80% of what appeared to be Cbl by microbiologic assay in spirulina was in fact analogues of Cbl. According to Herberts assumption, both the Chemilumi ACS B12 assay and RIDA using the intrinsic factor measures only genuine Cbl. On the other hand, another method, microbiological assay using E. coli 215, is known to measure the total of Cbl and its analogues. In our results, the values for Cbl (and analogues) obtained by the competitive binding assays coincided well with those obtained by the microbiological assay for raw asakusanori but higher values were obtained by the microbiological assay method for the dried asakusanori. This suggested that dried asakusanori contained more Cbl analogues than raw asakusanori. As to binding of Cbl analogues to an intrinsic factor, however, Kolhouse and Allen reported that some of their artificially prepared analogues of Cbl bound an intrinsic factor with affinity similar to that of genuine Cbl [16]. The results of van den Berg et al suggested the presence of Cbl analogues in nori and spirulina preparations that were ineffective in the human but bound to a porcine intrinsic factor, and therefore were mistaken for Cbl by RIDA [17]. Gimsing and Beck argued that determination of Cbl analogues by the difference of values obtained by RIDA using haptocorrin and an intrinsic factor as binders should be looked upon as a qualitative guess rather than a quantitative guess [18]. Therefore, our finding that the microbiological assay gave higher Cbl values than the competitive binding assays using an intrinsic factor may not, by itself, necessarily substantiate that the dried asakusanori contained a large proportion of Cbl analogues. However, other experiments, i.e. the analysis of Cbl components by paper chromatography and the determination of urinary methylmalonic acid excretion in this study gave pararell findings. Let us concentrate our attention on the effect of Cbl analogues on Cbl nutriture. Cbl analogues derived from Cbl in multivitamin-mineral pills inhibited Cbl-dependent enzyme methylmalonyl-Co A mutase when injected parenterally [19]. Stabler et al synthesized 16 Cbl analogues and administered them to rats [20]. Some Cbl analogues decreased liver methylmalonyl Co A mutase and methionine synthetase activities and increased serum methylmalonic acid and homocysteine concentrations. Haptocorrin, which can bind both Cbl and Cbl analogues, pre-

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vents the absorption of Cbl analogues in the intestine. That is, one of the physiological roles of haptocorrin is to prevent free Cbl analogues from binding to the receptor for the intrinsic factor-Cbl in the gut [21], otherwise analogues are absorbed via the receptor. In spite of this mechanism, some Cbl analogues are absorbed from the gastrointestinal tract and inhibit the Cbl-dependent enzyme activity as shown here. This may be the reason why Cbl analogues in dried asakusanori cause a worsening of Cbl nutriture. On the other hand, Suzuki [23] reported that no statistically significant difference was found between 6 vegan children aged 7 to 14 who had been living on a vegan diet from 4 to 10 years and an age-matched control group with respect to their serum Cbl levels and hematological indexes (red blood cell count, hematocrit, hemoglobin, etc.). He suggested that consumption of nori may keep vegans from suffering from Cbl deficiency. It is interesting that urinary methylmalonic acid increased when volunteers were given large amount of dried nori in our present experiments. The content of Cbl homologues in 40 g of dried asakusanori was on the average 5.53 g, as calculated from Table I. The content of Cbl analogues was calculated, 3. 62 g, as 65.4% of Cbl homologues are comprised of Cbl analogues in dried asakusanori as shown in Figure 2. The 3.62 g of Cbl analogues is less than 0.1% of the total Cbl stock in the normal human body [23]. That methylmalonic acid generation occurs with such a slight amount of Cbl analogue intake means very effective inhibition of methylmalonyl Co A mutase by Cbl analogues and higher affinity of the enzyme for Cbl analogues than for Cbl. The effect of Cbl analogues of dried asakusanori on methylmalonyl Co A mutase activity would be an interesting subject for future study. Let us now look at the content of Cbl analogues from a different point of view. It is thought that only Cbls that are bound to transcobalamin in plasma [24] are availabe for cells. The normal serum Cbl concentration is 500 pg/ml [25]. The average total blood volume of adults with a body weight of 60 kg is calculated to be 2.95 l because total blood weight equals 1/13 of body weight and the specific gravity of the blood is 1.056. The total Cbl content in the blood of adults is thus calculated to be 1.47 g. The 3.62 g of Cbl analogues ingested from dried asakusanori is 2.5 times greater than Cbl in blood and seems sufficient to affect Cbl nutriture even if the affinity of the enzyme for Cbl analogues is of the same order as for genuine Cbl. The identification of Cbl analogues in dried asakusanori and their effect on methylmalonyl Co A mutase are the next subjects for investigation. In our report, we have shown that 65% of the Cbl homologues were Cbl analogues in dried nori, which is a typical edible form of asakusanori, and the analogues con-

tained in it worsened Cbl nutriture. However, it is not necessary for us to avoid eating dried asakusanori because in the investigations of Dagnelie et al [5] or Rauma et al [3]. Cbl-deficient children or strict uncooked vegans were given a large amount of nori. In addition, in our present study 40 g of dried asakusanori correspond to twenty nori sheets of ordinary size, which is far beyond our routine intake. Urinary methylmalonic acid in these subjects was at the most less than 13 of 4.3 mol/mmol of creatinine which was the lower limit for diagnosis of definite Cbl deficiency. Usually raw asakusanori was eaten after being dispersed in hot water for a short time or was put into hot miso (soy bean paste) soup. Such a brief heating process does not matter because the Cbl content of raw asakusanori is almost completely retained after heat extraction at 90C for 30 min [4, 26]. In conclusion, it is not safe for Cbl-deficient people to eat dried asakusanori for the purpose of getting Cbl. However, raw asakusanori is an excellent source of genuine Cbl as a vegetable food.

Acknowledgements
This study was supported in part by a grant from the Hokkaido Food Science and Technology Promotion Foundation.

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Keiko Yamada Department of Liberal Arts and Sciences School of Health Sciences Sapporo Medical University South-1 and West-17, Chuo-ku, Sapporo 060-8556, Japan

Int. J. Vitam. Nutr. Res., 69 (6), 1999, Hogrefe & Huber Publishers