Effect of Ascorbic Acid on the Degradation of Cyanocobalamin and Hydroxocobalamin in Aqueous Solution: A Kinetic Study

The degradation kinetics of 5 × 10⁻⁵ M cyanocobalamin (B₁₂) and hydroxocobalamin (B_12b) in the presence of ascorbic acid (AH₂) was studied in the pH range of 1.0–8.0. B₁₂ is degraded to B_12b which undergoes oxidation to corrin ring cleavage products. B_12b alone is directly oxidized to the ring cleavage products. B₁₂ and B_12b in degraded solutions were simultaneously assayed by a two-component spectrometric method at 525 and 550 nm without interference from AH₂. Both degrade by first-order kinetics and the values of the rate constants at pH 1.0–8.0 range from 0.08 to 1.05 × 10⁻⁵ s⁻¹ and 0.22–7.62 × 10⁻⁵ s⁻¹, respectively, in the presence of 0.25 × 10⁻³ M AH₂. The t_1/2 values of B₁₂ and B_12b range from 13.7 to 137.5 h and 2.5–87.5 h, respectively. The second-order rate constants for the interaction of AH₂ with B₁₂ and B_12b are 0.05–0.28 × 10⁻² and 1.10–30.08 × 10⁻² M⁻¹ s⁻¹, respectively, indicating a greater effect of AH₂ on B_12b compared to that of B₁₂. The k_obs–pH profiles for both B₁₂ and B_12b show the highest rates of degradation around pH 5. The degradation of B₁₂ and B_12b by AH₂ is affected by the catalytic effect of phosphate ions on the oxidation of AH₂ in the pH range 6.0–8.0.KEY WORDS: ascorbic acid, cyanocobalamin, degradation, hydroxocobalamin, kinetics, two-component spectrometry     

Vitamin B<Subscript>12</Subscript> as a carrier for targeted platinum delivery: in vitro cytotoxicity and mechanistic studies

It is attractive to use vitamin B₁₂ as a carrier for targeted delivery of cytotoxic agents such as platinum complexes owing to the high demand for vitamin B₁₂ by fast proliferating cells. The basic {B₁₂–CN–Pt^II} conjugates are recognized by intracellular enzymes and converted to coenzyme B₁₂ in an enzymatic adenosylation assay. The reductive adenosylation of {B₁₂–CN–Pt^II} conjugates leads to the release of the Pt^II complexes; thus, {B₁₂–CN–Pt^II} conjugates can be considered as prodrugs. It is important not only to elucidate the activity of the cisplatin–B₁₂ conjugates, but also to understand the mode of action on a molecular level. Chemical reduction of {B₁₂–CN–Pt^II} conjugates with cobaltocene yielded cob(II)alamin and induced release of the corresponding Pt^II species. Kurnakov tests and coordination of 2′-deoxyguanosine or GMP to the released Pt^II complexes allowed isolation and characterization of Pt^II complexes as released during enzymatic adenosylation. The biological activity of these Pt^II complexes was evaluated. Since the cleaved Pt^II complexes show cytotoxicity, the {B₁₂–CN–Pt^II} conjugates can be used for specific targeting of cancer cells and therapeutic drug delivery. Preliminary in vitro cytotoxicity studies indicated lower activity (IC₅₀ between 8 and 88 μM) than found for pure cisplatin. Since active transport and receptor-mediated uptake limits the intracellular {B₁₂–CN–Pt^II} concentration, comparison with pure cisplatin is of limited use. We could show that the Pt^II complexes cleaved from B₁₂ exerted a cytotoxicity comparable to that of cisplatin itself. Cytotoxicity studies in vitamin B₁₂ free media showed a dependence on the addition of transcobalamin II for B₁₂–Pt(II) conjugates.     

Metformin increases liver accumulation of vitamin B12 – An experimental study in rats

Aims/hypothesis

Patients treated with metformin exhibit low levels of plasma vitamin B₁₂ (B₁₂), and are considered at risk for developing B₁₂ deficiency. In this study, we investigated the effect of metformin treatment on B₁₂ uptake and distribution in rats.

Methods

Sprague Dawley rats (n = 18) were divided into two groups and given daily subcutaneous injections with metformin or saline (control) for three weeks. Following this, the animals received an oral dose of radio-labeled B₁₂ (⁵⁷[Co]-B₁₂), and urine and feces were collected for 24 h. Plasma, bowel content, liver, and kidneys were collected and analyzed for B₁₂, unsaturated B₁₂-binding capacity, and ⁵⁷[Co]-B₁₂.

Results

Three weeks of metformin treatment reduced plasma B₁₂ by 22% or 289 [47-383] pmol/L (median and [range]) (p = 0.001), while no effect was observed on unsaturated B₁₂-binding capacity. Compared with controls, the amount of B₁₂ in the liver was 36% (p = 0.007) higher in metformin-treated rats, while the B₁₂ content in the kidney was 34% (p = 0.013) lower. No difference in the total amount of absorbed ⁵⁷[Co]-B₁₂ present in the tissues and organs studied was found, suggesting that metformin has no decreasing effect on the B₁₂ absorption.

Conclusions/interpretation

These results show that metformin treatment increases liver accumulation of B₁₂, thereby resulting in decreases in circulating B₁₂ and kidney accumulation of the vitamin. Our data questions whether the low plasma B₁₂ observed in patients treated with metformin reflects impaired B₁₂ status, and rather suggests altered tissue distribution and metabolism of the vitamin.     

Rb2[B12(OH)12]·2H2O and Rb2[B12(OH)12]·2H2O2: Hydrate and perhydrolate of rubidium dodecahydroxo-closo-dodecaborate

The orthorhombically crystallizing salts Rb₂[B₁₂(OH)₁₂]·2H₂O (a = 1576.81(9), b = 813.08(5), c = 1245.32(7) pm) and Rb₂[B₁₂(OH)₁₂]·2H₂O₂ (a = 1616.54(9), b = 814.29(5), c = 1260.12(7) pm) could be prepared from Rb₂[B₁₂H₁₂] and hydrogen peroxide. Both crystal structures were determined by X-ray single crystal diffraction and refined in the space group Cmce. They are not isostructural to the other compounds containing icosahedral dodecahydroxo-closo-dodecaborate dianions [B₁₂(OH)₁₂]²⁻ and potassium, rubidium or cesium cations already known to literature, but both title compounds crystallize quasi-isotypically exhibiting Rb⁺ cations in 10-fold oxygen coordination. The hydrogen peroxide adduct (Rb₂[B₁₂(OH)₁₂]·2H₂O₂) is explosive on shock and heat, while the hydrate (Rb₂[B₁₂(OH)₁₂]·2H₂O) is not.     

The β-ligands

The 5-hydroxybenzimidazolylcobamide (B₁₂-HBI) derivatives from Methanosarcina barkeri were isolated. SO₃^2?, CN^?, H₂O, NH₃ and CH₃^? were identified as β-ligands. Two B₁₂-HBI compounds with unidentified β-ligands were found, of which one constituted a major part of the corrinoid content. 5′-Deoxyadenosyl was found as a β-ligand of a corrinoid without α-ligand. Biosynthesis of CH₃B₁₂HBI was observed in cell-free extracts and depended on methanol and ATP.     

Towards the synthesis of light-stable coenzyme B12 analogs

The organometallic complex coenzyme B₁₂ (adenosyl cobalamin, AdoCbl) is not only an essential coenzyme in many biochemical reactions of most if not all living organisms but has lately been shown to play a crucial role in the regulation of B₁₂ related genes. As a consequence, coenzyme B₁₂ has been a target of intense research. However, the investigations of AdoCbl have often been hampered due to its high light-sensitivity leading to decomposition of the compound within a few seconds. Here, we describe a strategy to synthesize more light-stable coenzyme B₁₂ analogs, which show similar steric properties as adenosyl cobalamin. The synthesis, structural characterization as well as the pH dependent “base-on/base-off” behavior of cyanide bridged vitamin B₁₂ conjugates with either a cis-[(NH₃)₂Pt]²⁺ or an [enPt]²⁺ moiety, leading to cis-[(NH₃)₂PtCl-vitB₁₂]⁺ (1) and [enPtCl-vitB₁₂]⁺ (2) are reported. The subsequent reaction of cis-[(NH₃)₂PtCl-vitB₁₂]⁺ with the model nucleobase 9-methyladenine leads to the corresponding adduct, where the adenine moiety is coordinated to the Pt²⁺ center either via N1 or N7. This compound is light-stable and harbors the adenine moiety in the same distance of 5 Å above the corrin plane as present in the highly light-sensitive adenosyl cobalamin.     

The effect of vitamin B12 and biotin on the metabolism of vitamin B12 in biotin-deficient rats

1. The effect of the administration of vitamin B₁₂ and biotin on the metabolic pattern of vitamin B₁₂ in biotin-deficient rats was studied. 2. No significant changes in the absorption and excretion of orally administered [⁵⁸Co]vitamin B₁₂ were noted either in vitamin B₁₂-treated and or in biotin-fed rats. A significant decrease of the uptake of orally given [⁵⁸Co]vitamin B₁₂ was observed in the liver and kidneys of biotin-treated rats, whereas an increase of uptake in the kidneys of vitamin B₁₂-treated rats was noted as compared with biotin-deficient animals. 3. No significant difference in the excretion of radioactivity was noted between biotin deficient and biotin-fed rats when [⁵⁸Co]vitamin B₁₂ was administered by injection. A small decrease was observed in vitamin B₁₂-treated rats. The retention of injected [⁵⁸Co]vitamin B₁₂ by major organs of biotin-treated rats was lower than that of biotin-deficient rats. A lower content of [⁵⁸Co]vitamin B₁₂ was also detected in the organs, with the exception of the kidneys, of vitamin B₁₂-treated rats. 4. These results are discussed in terms of an interrelationship between biotin and vitamin B₁₂.     

Transport of vitamin B12 into mouse leukemia cells.

Transport of [⁵⁷Co]cyanocobalamin (vitamin B₁₂) into L1210 murine leukemia cells, mediated by transcobalamin-II, is a biphasic process. The primary step, which occurs very rapidly (within the first minute), appears to involve binding of the B₁₂-transcobalamin complex to the external membrane of the cells; at 37 °C and pH 7.3, apparent K_m and V values are 180 pM and 5.9 pmol/min/10⁹ cells, respectively. This step is relatively insensitive to temperature, has an apparent pK_a of 6.0, and is inhibited by EDTA; Ca²⁺ or Mg²⁺ can reverse the inhibition. The slower secondary step appears to involve translocation of the vitamin into the cell; V for this step is 0.4 pmol/min/10⁹ cells. Temperature and pH optima are 30 ° and 6.5?7.0, respectively. This step is stimulated by glucose and inhibited by cyanide, arsenite, arsenate, p-chloromercuriphenylsulfonate and dinitrophenol. Labeled B₁₂ in an amount corresponding to that taken up in the initial step can be released by: (a) resuspending the cells in a B₁₂-free medium; (b) addition of unlabeled B₁₂-transcobalamin-II; and (c) addition of EDTA. The material released is chromatographically and functionally identical with B₁₂-transcobalamin-II.     

Hooked to vitamin B12 since 1955: A historical perspective

In our pioneering work in 1956, two binders of vitamin B₁₂ (B₁₂) alias cobalamin (Cbl) were identified in gastric juice, S with slow electrophoretic mobility, a 70 kD protein with intrinsic factor (IF) activity and another rapid (R), not IF active but probable digestion product. Numerous sources contained a protein immunologically identical to R (haptocorrin, Hc). Another IF-active component (I) was found. Isoelectric focusing showed that S, I and R were assemblies of “isoproteins” with different pI's due to varying glycosidation. Isolation of S, I and R in microquantities was achieved in 1962 using a series of ion exchange chromatographies and gel filtration. Ponderable products were obtained in 1965–1966. The B₁₂-IF complex was a dimer, contained 13% carbohydrate and showed a different absorption spectrum than B₁₂. Using the Schilling test, B₁₂ absorption was shown to require Ca⁺⁺, bound in vitro to the ileal receptor and IF, but most of Ca⁺⁺ could be removed with sialidase. The receptor–substrate complex contained Ca⁺⁺ and carbohydrate. The purified receptor was shown to contain two main subunits. The Imerslund–Gräsbeck syndrome was discovered 1958–1960; it is caused by mutations in either of two genes, cubilin or amnionless, which form the multiligand receptor cubam. Testicular biopsies during and after B₁₂-treated deficiency showed remarkable improvement after therapy. Studies of the turnover of radioactive B₁₂ revealed biliary and fecal excretion, enterohepatic circulation and allowed calculation of biological half-life and daily need. The B₁₂ coenzymes largely behaved like B₁₂. To study whether radiocobalt in B₁₂ was representative of the rest of the B₁₂ molecule, ³²P and ⁵⁷Co labeled hydroxocobalamins were biosynthesized and shown to behave identically when given simultaneously to rats. The complex metabolism of B₁₂ explains the pathogenesis of B₁₂ deficiencies. Some of its mechanisms are not restricted to B₁₂, e.g. the endocytosis of B₁₂-IF also applies to other macromolecules.     

Studies of the ribosome-associated vitamin B12S adenosylating enzyme of Lactobacillus leichmannii.

The ribosomes of Lactobacillus leichmannii (ATCC 7830) are the loci of an enzyme system that converts vitamin B₁₂ (cyanocobalamin, CN-Cbl) to adenosylcobalamin (AdoCbl) in two steps, reduction of B_12a (Co III) to B_12s (Co I) by B_12a reductase and the adenosylation of B_12s to AdoCbl by an adenosylating enzyme. The vitamin B₁₂ reductase, as in other organisms, is unstable. Adenosylating enzyme, however, is readily demonstrable. Reported experiments deal primarily with that enzyme. Evidence of the association between ribosomes and adenosylating enzyme was found in sucrose density gradient analyses. Intact, washed ribosomes yielded an enzyme activity profile that coincided with the ultraviolet maximum of 70S reference ribosomes. When intact ribosomes were exposed to 2.5 m CsCl so that 70% of ribosomal protein was recoverable in the 144,000g supernatant fraction and >90% of RNA was in the pellet, adenosylating enzyme was found in the supernatant fraction. “Stripped” ribosomes had low levels of enzyme activity and could reassociate with free enzyme protein. Stripped ribosomes remained competent in protein synthesis. Hence, adenosylating enzyme is not an integral ribosome component. Partially purified ribosome-associated B_12s adenosylating enzyme has requirements for vitamin B_12s, ATP, and Mn²⁺, though Mn²⁺ could be partially replaced by Mg²⁺. Isotopic studies showed that ATP is the source of the adenosyl moiety of AdoCbl and that inorganic tripolyphosphate is a reaction product. Substantial adenosylating activity is associated with ribosomes only in the vitamin B₁₂-requiring lactobacilli, L. delbrueckii (ATCC 9649) and L. leichmannii. Surprisingly, L. casei (ATCC 9595) ribosomes displayed a measurable, if low, level of activity. L. acidophilus (ATCC 11506) ribosomes had no detectable activity. The bulk of the activity in Clostridium tetanomorphum (ATCC 3606) and Propionibacterium shermannii (ATCC 9614) is in the 144,000g supernatant fraction. Ribosomes from animal cells (liver, reticulocytes, and Ehrlich ascites tumor) were without detectable activity.     

Vitamin analysis of five planktonic microalgae and one macroalga

Vitamin analysis was carried out on five microalgae used in aquaculture:Tetraselmis suecica, Isochrysis galbana, Pavlova lutheri, Skeletonema costatum andChaetoceros calcitrans and one macroalga,Sargassum muticum, which is invasive on the Atlantic shores of France. Both liposoluble (provitamin A, E, K) and hydrosoluble (B₁, B₂, B₆, B₁₂, C, PP) vitamins were quantified. For most of them, greater amounts were obtained in the algal products than in the usual sources. On a dry weight basis,Tetraselmis suecica contained 4280 μg g^?1 provitamin A and 6323 μg g^?1 vitamin E,Pavlova lutheri 1162 μg g^?1 vitamin B₁₂ and 837 μg g^?1 vitamin C,Isochrysis galbana 2690 μg g^?1 vitamin PP and 183 μg g^?1 vitamin B₆, andSkeletonema costatum 710 μg g^?1 vitamin B₁.     

Influence of Zn<Superscript>2+</Superscript>, Co<Superscript>2+</Superscript> and dimethylbenzimidazole on vitamin B<Subscript>12</Subscript> biosynthesis by <Emphasis Type="Italic">Pseudomonas denitrificans</Emphasis>

Previous research has confirmed that cobalt ion and dimethylbenzimidazole (DMBI) are the precursors of vitamin B₁₂ biosynthesis, and porphobilinogen synthase (PBG synthase) is a zinc-requiring enzyme. In this paper, the effects of Zn²⁺, Co²⁺ and DMBI on vitamin B₁₂ production by Pseudomonas denitrificans in shake flasks were studied. Present experimental results demonstrated that the addition of the above mentioned three components to the fermentation medium could significantly stimulate the biosynthesis of vitamin B₁₂. The concentrations of zinc sulphate, cobaltous chloride and DMBI in the fermentation medium were further optimized with rotatable orthogonal central composite design and statistical analysis by Data Processing System (DPS) software. As a result, vitamin B₁₂ production was increased from 69.36 ± 0.66 to 78.23 ± 0.92 μg/ml.     

Mouse transcobalamin II: Biosynthesis and uptake by L-929 cells

Mouse fibroblast (L-929) cells, in culture, synthesized and secreted into the growth medium a vitamin B₁₂-binding substance which was identical to mouse transcobalamin II (TC II) as judged by the following criteria: (i) gel filtration on Sephadex G-200, (ii) ion-exchange chromatography on DEAE-cellulose and CM-cellulose, and (iii) the ability to facilitate cellular B₁₂ uptake by L-929 cells. The secretion of mouse fibroblast binder was blocked by cycloheximide and puromycin; and in both cases the cells' ability to secrete this binder was partially restored when the inhibitor was removed. Within 30 h after the cells were exposed to [⁵⁷Co]B₁₂ bound to mouse serum TC II (M_r ~ 38,000) the [⁵⁷Co]B₁₂ was bound to a large molecular weight intracellular binder (M_r ~ 120,000) which was not released into the culture medium. During this same incubation period, the cells released free [⁵⁷Co]B₁₂ and [⁵⁷Co]B₁₂ bound to a protein which had the same elution volume as mouse serum TC II on Sephadex G-200.     

Vitamin B(12) incorporated with multiwalled carbon nanotube composite film for the determination of hydrazine

Electrochemically active composite film containing multiwalled carbon nanotubes (MWCNTs) and vitamin B₁₂ was synthesized on glassy carbon, gold, and indium tin oxide electrodes by the potentiodynamic method. The presence of MWCNTs in the composite film (MWCNT–B₁₂) modified electrode mediates vitamin B₁₂’s redox reaction, whereas vitamin B₁₂’s redox reaction does not occur at bare electrode. The electrochemical impedance spectroscopy studies reveal that MWCNTs present in MWCNT–B₁₂ film enhance electron shuttling between the reactant and electrode surface. The surface morphology of bare electrode, MWCNT film. and MWCNT–B₁₂ composite film was studied using atomic force microscopy, which reveals vitamin B₁₂ incorporated with MWCNTs. The MWCNT–B₁₂ composite film exhibits promising enhanced electrocatalysis toward hydrazine. The electrocatalysis response of hydrazine at MWCNT film and MWCNT–B₁₂ composite film was measured using cyclic voltammetry and amperometric current–time (i–t) curve techniques. The linear concentration range of hydrazine obtained at MWCNT–B₁₂ composite film using the i–t curve technique is 2.0 μM–1.95 mM. Similarly, the sensitivity of MWCNT–B₁₂ composite film for hydrazine determination using the i–t curve technique is 1.32 mA mM⁻¹ cm⁻², and the hydrazine’s limit of detection at MWCNT–B₁₂ composite film is 0.7 μM.     

Studies on the mechanism of formation of the 5S,12S-dihydroxy-6,8,10,14(E,Z,E,Z)-icosatetraenoic acid in leukocytes

Incubation of peripheral blood leukocytes with arachidonic acid (and ionophore A23187) led to the formation of leukotriene B₄, Δ⁶-trans-leukotriene B₄, Δ⁶-trans-12-epi-leukotriene B₄, 5-hydroxy-icosatetraenoic acid, 12-hydroxy-icosatetraenoic acid and of 5S,12S-dihydroxy-6,8,10,14-(E,Z,E,Z)-icosatetraenoic acid (5S,12S-DiHETE). Incubation of leukocytes with leukotriene A₄ resulted in the formation of leukotriene B₄ and of its two Δ⁶-trans-isomers but not of the 5S,12S-DiHETE. ¹⁸O₂ labeling experiments have shown that the hydroxyl groups at C5 and C12 in the 5S,12S-DiHETE are derived from molecular oxygen. The tetraacetylenic analog of arachidonic acid was found to be a potent inhibitor of the formation of the 5S,12S-DiHETE whereas it potentiated the synthesis of the 5-hydroxy acid and of leukotriene B₄. Addition of the 12-hydroxy-icosatetraenoic acid to leukocytes, or of the 5-hydroxy-icosatetraenoic acid to a suspension of platelets caused the formation of the 5S,12S-DiHETE. It is concluded that the 5S,12S-DiHETE is not derived from leukotriene A₄ but is a product of the successive reactions of arachidonic acid with two lipoxygenases of different positional specificities.     

Enrichment of some B-vitamins in plants with application of organic fertilizers

A review of the literature showed that plants grown with organic fertilizers often contain higher concentrations of vitamins B₁ (thiamin) and B₁₂ (cyanocobalamin) as compared with plants grown with inorganic fertilizers. Since plant roots were recently shown to be able to absorb B₁ and B₁₂, it was thus suspected that organic fertilizers (such as manure of diverse sources or sewage sludges which often contain relatively high concentrations of several vitamins) introduce additional vitamins into the soil which in turn leads to increased vitamins in the plants. This possibility was studied by measuring the B₁₂ content in the seeds of soybean and barley and in the leaves of spinach plants grown in soils amended with pure B₁₂ or cow dung (which is naturally rich in B₁₂). The addition of pure B₁₂ or cow dung did not alter the B₁₂ content in the soybean seeds but significantly increased that in the barley kernels and in the spinach leaves. For example, the addition of cow dung at the rate of 10 g kg^–1 increased the B₁₂ content in barley kernels by more than threefold (from 2.6 to 9.1 ng g^–1 DW) and in spinach leaves by close to twofold (from 6.9 to 17.8 ng g^–1 DW). Long-term addition of organic fertilizers to the soil also significantly increased the soil content of this vitamin. Since plants cannot synthesize B₁₂ and thus plant foods are normally fully devoid of (or have very low concentrations of) this vitamin, the finding that plants grown with organic fertilizers may contain relatively higher concentrations of this vitamin may have nutritional consequences in that the consumption of these plants by humans would inadvertently increase their intake of this vitamin. This may be of special benefit to people living by choice or by necessity on strict vegetarian diets who are known to be in danger of B₁₂ deficiency.     

Enhancement of chemiluminescence for vitamin B12 analysis

In the current article, chemiluminescence (CL) from the vitamin B₁₂ and luminol reaction was studied under alkaline conditions to develop a sensitive analytical method for vitamin B₁₂ using the carbonate enhancement effect. The method was successfully applied to the determination of vitamin B₁₂ in vitamin B₁₂ tablets, multivitamin capsules, and vitamin B₁₂ injections. Experimental parameters were optimized, including luminol concentration, urea-hydrogen peroxide (urea-H₂O₂) concentration, effect of pH, and sequence of addition of reactants for obtaining maximum CL, which was not explored previously. The limit of detection was 5 pg/ml, and the linear range was 10 pg/ml to 1 μg/ml with a regression coefficient of R² = 0.9998. The importance of these experimental parameters and the carbonate enhancement effect is discussed based on the knowledge of the mechanism of oxidation of luminol and decomposition of urea-H₂O₂ in the presence of vitamin B₁₂. Extraction of vitamin B₁₂ was carried out, and the observed recovery was 97-99.2% with a relative standard deviation in the range of 0.30-1.09%. The results obtained were compared with those of the flame atomic absorption spectrometry method.     

Inhibitory action of soluble elastin on thromboxane B2 formation in blood platelets

Soluble elastin, prepared from insoluble elastin by treatment with oxalic acid or elastase, was found to inhibit the formation of thromboxane B₂ both from [1-¹⁴C]arachidonic acid added to washed platelets and from [1-¹⁴C]arachidonic acid in prelabeled platelets on stimulation with thrombin. In both systems, the formation of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) was accelerated. Oxalic acid-treated soluble elastin st 1 and 10 mg/ml inhibited the formation of thromboxane B₂ from exogenously supplied arachidonic acid 21 and 59%, respectively, and the formation of thromboxane B₂ in prelabeled platelets stimulated by thrombin 44 and 94%, respectively. These concentrations of elastin increased the formation of 12-HETE from exogenously supplied arachidonic acid about 3.4- and 7.3-times, respectively. Almost all the added arachidonic acid was converted to metabolites. In prelabeled platelets, soluble elastin at 1 and 10 mg/ml increased the formation of 12-HETE stimulated by thrombin about 1.3- and 2.8-times, respectively, and inhibited the thrombin-induced total productions of thromboxane B₂ (12-hydroxy-5,8,10-heptadecatrienoic acid (12-HETE) and free arachidonic acid by 26 and 25%, respectively. Elastase-treated digested elastin also inhibited the formation of thromboxane B₂ and stimulated the formation of 12-HETE in prelabeled platelets stimulated by thrombin. This inhibitory action of elastin was not replaced by desmosine. The level of cAMP in platelets was not affected by soluble elastin. Soluble elastin was also found to inhibit platelet aggregation induced by thrombin. However, the inhibitory action of soluble elastin on platelet aggregation cannot be explained by inhibition of thromboxane B₂ formation by the elastin.     

UNEXPECTED RESPONSE TO VITAMIN B12 OF DOMINANT CENTRIC DIATOMS FROM THE SPRING BLOOM IN THE GULF OF MAINE (NORTHEAST ATLANTIC OCEAN)1,2

Twelve clones (seven species) representative of centric diatoms dominant in the spring phytoplankton bloom in the Gulf of Maine were isolated and rendered axenic. Genera included were Thalassiosira, Porosira and Chaetoceros. Unlike most centric diatoms studied previously, none of these has an absolute requirement for vitamin B_12. However, B₁₂ (5 ng.l^-1) stimulated growth of most clones by eliminating or reducing the lag phase and increasing the growth rate. Bloom population densities developed 4–54 days earlier with B₁₂ present. Several clones grown with B₁₂ removed more than 80% of the vitamin from the medium. When grown in vitamin-free medium the cells put 0.01–0.7 ng.l^-1 B₁₂ into the medium. We conclude that vitamin B₁₂ is of ecological significance even though the requirement for it is not absolute.     

Vitamin B12 Content of Circulating Leukocytes as an Aid in the Differentiation of the Acute Leukemias

From 25 patients with acute leukemia 116 specimens of leukocytes were assayed microbiologically for total vitamin B₁₂ to determine if variation in vitamin B₁₂ content would help in differentiating the acute leukemias. The mean cell vitamin B₁₂ levels (μμg./10⁸ cells) in the different types of leukemia were: lymphoblastic 464, myeloblastic 1058 and monocytic 200. Cell vitamin B₁₂ levels above the normal range (100-800 μμg./10⁸ cells) are suggestive of myeloblastic leukemia. The only elevated cell vitamin B₁₂ levels comparable to those found in myeloblastic leukemia were in reticulum cell leukemia, and this type of leukemia was not difficult to diagnose morphologically. Blast cells contained more vitamin B₁₂ than mature cells of the same series; there was a significant positive correlation between the percentage of blast cells and cell levels of total vitamin B₁₂ in both lymphoblastic and myeloblastic leukemia.