Nitric oxide interactions with cobalamins: biochemical and functional consequences

Nitric oxide (NO) is a paramagnetic gas that has been implicated in a wide range of biologic functions. The common pathway to evoke the functional response frequently involves the formation of an iron- nitrosyl complex in a target (heme) protein. In this study, we report on the interactions between NO and cobalt-containing vitamin B12 derivatives. Absorption spectroscopy showed that of the four Co(III) derivatives (cyanocobalamin [CN-Cbl], aquocobalamin [H2O-Cbl], adenosylcobalamin [Ado-Cbl], and methylcobalamin [MeCbl]), only the H2O- Cbl combined with NO. In addition, electron paramagnetic resonance spectroscopy of H2O-Cbl preparations showed the presence of a small amount of Cob-(II)alamin that was capable of combining with NO. The Co(III)-NO complex was very stable, but could transfer its NO moiety to hemoglobin (Hb). The transfer was accompanied by a reduction of the Co(III) to Co(II), indicating that NO+ (nitrosonium) was the leaving group. In accordance with this, the NO did not combine with the Hb Fe(II)-heme, but most likely with the Hb cysteine-thiolate. Similarly, the Co(III)-NO complex was capable of transferring its NO to glutathione. Ado-Cbl and Me-Cbl were susceptible to photolysis, but CN- Cbl and H2O-Cbl were not. The homolytic cleavage of the Co(III)-Ado or Co(III)-Me bond resulted in the reduction of the metal. When photolysis was performed in the presence of NO, formation of NO-Co(II) was observed. Co(II)-nitrosyl oxidized slowly to form Co(III)-nitrosyl. The capability of aquocobalamin to combine with NO had functional consequences. We found that nitrosylcobalamin had diminished ability to serve as a cofactor for the enzyme methionine synthase, and that aquocobalamin could quench NO-mediated inhibition of cell proliferation. Our in vitro studies therefore suggest that interactions between NO and cobalamins may have important consequences in vivo.     

The pharmacokinetic and pharmacodynamic properties of biphasic insulin Aspart 70 (BIAsp 70) are significantly different from those of biphasic insulin Aspart 30 (BIAsp 30).

AIMS: To evaluate the pharmacokinetic and pharmacodynamic properties of two different formulations of premixed analogue (mixed biphasic insulin aspart [BIAsp]), BIAsp 30 (30 % soluble insulin aspart [IAsp] and 70 % protaminated IAsp) and BIAsp 70 (70 % soluble IAsp and 30 % protaminated IAsp), in patients with type 1 diabetes using a 12-hour euglycaemic clamp technique. METHODS: In this randomised, double-blind, two-period crossover trial, 27 patients with type 1 diabetes received 7 days of treatment with BIAsp 30 three times daily (TID) and 7 days of treatment with BIAsp 70 TID, with a 2 - 6 week washout period between treatment periods. At the start (day 1) and end (day 8) of each treatment period, 12-hour serum IAsp profiles and glucose infusion rate (GIR) profiles were determined during an overnight euglycaemic clamp following subcutaneous (sc) administration of a single 0.3 U/kg dose of trial insulin. All pharmacokinetic and pharmacodynamic endpoints were derived from individual serum IAsp and GIR profiles. RESULTS: The larger fraction of soluble IAsp in BIAsp 70 compared with BIAsp 30 resulted in greater metabolic effect during the initial post-dosing phase (0 - 6 hours) and decreased activity during the late phase (6 - 12 hours). On day 8, AUC (GIR 0 - 6 hours) was 16 % greater for BIAsp 70 than for BIAsp 30 (p = 0.016) and AUC (GIR 6 - 12 hours) was 90 % (p < 0.001) greater for BIAsp 30 relative to BIAsp 70, reflecting the increased proportion of intermediate-acting (protamine co-crystallised) IAsp in BIAsp 30. Overall metabolic effect (AUC (GIR 0 - 12 hours)) was similar for both insulin formulations on day 8 (Ratio, BIAsp 30:BIAsp 70 = 1.04, p = 0.538). Likewise, the larger fraction of soluble IAsp in BIAsp 70 compared with BIAsp 30 resulted in greater exposure to IAsp during the initial post-dosing phase (0 - 6 hours) and a smaller exposure to IAsp during the late phase (6 - 12 hours). On day 8, AUC (IAsp 0 - 6 hours) was 59 % (p < 0.001) greater for BIAsp 70 than for BIAsp 30, and AUC (IAsp 6 - 12 hours) was 61 % (p < 0.001) greater for BIAsp 30 than for BIAsp 70, reflecting the increased proportion of intermediate-acting (protamine co-crystallised) IAsp in BIAsp 30. However, the overall IAsp exposure (AUC 0 - 12 hours) on day 8 was significantly greater for BIAsp 70 than for BIAsp 30 (Ratio, BIAsp 30:BIAsp 70 = 0.73, p < 0.001). The GIR profiles on day 8 were similar to those on day 1. Serum IAsp profiles on day 8 showed a slight increase compared with day 1. CONCLUSIONS: The different proportions of soluble and protaminated IAsp result in differences in the pharmacokinetic and pharmacodynamic properties of BIAsp 30 and BIAsp 70. The pharmacokinetic and pharmacodynamic differences observed may allow for flexibility and individualised treatment regimens in patients with diabetes, while maintaining a limited number of daily injections.     

Abnormal pregnancy: early diagnosis by US and serum chorionic gonadotropin levels

Simultaneous sonography and quantitative serum human chorionic gonadotropin (HCG) levels from 126 women with threatened abortion were compared. Of 56 women with normal outcome, 39 (70%) had a gestation sac greater than or equal to 5 mm in mean sac diameter, and in each case the HCG level was 1,800 milli-international units (mIU/ml) or greater. The serum HCG levels strongly correlated with the gestation sac sizes to a mean sac diameter of 25 mm. Of 70 abnormal pregnancies, 31 demonstrated a gestation sac. Of these, 20 women (65%) had disproportionately low HCG levels relative to sac size, including 12 in whom the HCG level was less than 1,800 mIU/ml. One woman with an early molar pregnancy had a disproportionately elevated HCG level. Correlation of sonograms with a simultaneous measurement of serum HCG level is a useful method for evaluating threatened spontaneous abortion. A disproportionately low HCG level relative to gestation sac size is evidence for an abnormal pregnancy.     

Anti-IgM induces transforming growth factor-beta sensitivity in a human B-lymphoma cell line: inhibition of growth is associated with a downregulation of mutant p53

We wished to examine the role of transforming growth factor-beta (TGF- beta) in the regulation of human lymphoma cell growth. The RL cell line is an immunoglobulin M (IgM)+, IgD+ B lymphoma cell line, which does not constitutively express receptors for TGF-beta, and thus has lost the ability to respond to the inhibitory effects of TGF-beta. We demonstrate here that anti-Ig antibodies can efficiently upregulate the expression of TGF-beta receptors and promote sensitivity to growth inhibition by TGF-beta. Furthermore, because TGF-beta has been shown to function in late G1 of the cell cycle, we examined the ability of TGF- beta to modulate two tumor suppressor proteins known to be critical regulators of the G1/S transition, Rb and p53. Rb is a 105- to 110-kD phosphoprotein, which has been shown to maintain its growth suppressive function when it is found in the hypophosphorylated state. Wild-type p53 is a 53-kD phosphoprotein that appears to be important in preventing cell-cycle progression and promoting apoptosis in cells with DNA damage, whereas mutant p53 can overcome those functions. We show here that TGF-beta treatment of phorbol myristate acetate (PMA) or anti- Ig-activated RL cells results in growth inhibition through a dual effect on Rb and mutant p53. After TGF-beta treatment, we observe a predominance of Rb in the hypophosphorylated, growth suppressive form. In addition, we show a decrease in levels of mRNA and protein for mutant p53. We also show that, although these changes are sufficient to halt progression through the cell cycle, the cells do not appear to undergo extensive programmed cell death following 72 hours of TGF-beta treatment. Thus, although these lymphoma cells maintain the capacity to be negatively growth regulated by TGF-beta, the ability of TGF-beta to induce apoptosis must be independently controlled.