Enhanced absorption of insulin aspart as the result of a dispersed injection strategy tested in a randomized trial in type 1 diabetic patients.

OBJECTIVE: We investigated the impact of two different injection strategies on the pharmacokinetics and pharmacodynamics of insulin aspart in vivo in an open-label, two-period crossover study and verified changes in the surface-to-volume ratio ex vivo. RESEARCH DESIGN AND METHODS: Before the clinical trial, insulin aspart was injected ex vivo into explanted human abdominal skin flaps. The surface-to-volume ratio of the subcutaneous insulin depot was assessed by microfocus computed tomography that compared 1 bolus of 18 IU with 9 dispersed boluses of 2 IU. These two injection strategies were then tested in vivo, in 12 C-peptide-negative type 1 diabetic patients in a euglycemic glucose clamp (glucose target 5.5 ± 1.1 mmol/L) for 8 h after the first insulin administration. RESULTS: The ex vivo experiment showed a 1.8-fold higher mean surface-to-volume ratio for the dispersed injection strategy. The maximum glucose infusion rates (GIR) were similar for the two strategies (10 ± 4 vs. 9 ± 4; P = 0.5); however, times to reach maximum GIR and 50% and 10% of the maximum GIR were significantly reduced by using the 9 × 2 IU strategy (68 ± 33 vs. 127 ± 93 min; P = 0.01; 38 ± 9 vs. 49 ± 16 min; P < 0.01; 23 ± 6 vs. 30 ± 10 min; P < 0.05). For 9 × 2 IU, the area under the GIR curve was greater during the first 60 min (219 ± 89 vs. 137 ± 75; P < 0.01) and halved until maximum GIR (242 ± 183 vs. 501 ± 396; P < 0.01); however, it was similar across the whole study period (1,361 ± 469 vs. 1,565 ± 527; P = 0.08). CONCLUSIONS: A dispersed insulin injection strategy enhanced the effect of a fast-acting insulin analog. The increased surface-to-volume ratio of the subcutaneous insulin depot can facilitate insulin absorption into the vascular system.

Degradation of organic pollutants by methane grown microbial consortia.

Microbial consortia were enriched from various environmental samples with methane as the sole carbon and energy source. Selected consortia that showed a capacity for co-oxidation of naphthalene were screened for their ability to degrade methyl-tert-butyl-ether (MTBE), phthalic acid esters (PAE), benzene, xylene and toluene (BTX). MTBE was not removed within 24 h by any of the consortia examined. One consortium enriched from activated sludge ("AAE-A2"), degraded PAE, including (butyl-benzyl)phthalate (BBP), and di-(butyl)phthalate (DBP). PAE have not previously been described as substrates for methanotrophic consortia. The apparent Km and Vmax for DBP degradation by AAE-A2 at 20 degrees C was 3.1 +/- 1.2 mg l(-1) and 8.7 +/- 1.1 mg DBP (g protein x h)(-1), respectively. AAE-A2 also showed fast degradation of BTX (230 +/- 30 nmol benzene (mg protein x h)(-1) at 20 degrees C). Additionally, AAE-A2 degraded benzene continuously for 2 weeks. In contrast, a pure culture of the methanotroph Methylosinus trichosporium OB3b ceased benzene degradation after only 2 days. Experiments with methane mono-oxygenase inhibitors or competitive substrates suggested that BTX degradation was carried out by methane-oxidizing bacteria in the consortium, whereas the degradation of PAE was carried out by non-methanotrophic bacteria co-existing with methanotrophs. The composition of the consortium (AAE-A2) based on polar lipid fatty acid (PLFA) profiles showed dominance of type II methanotrophs (83-92% of biomass). Phylogeny based on a 16S-rRNA gene clone library revealed that the dominating methanotrophs belonged to Methylosinus/Methylocystis spp. and that members of at least 4 different non-methanotrophic genera were present (Pseudomonas, Flavobacterium, Janthinobacterium and Rubivivax).

Recombinant human serum amyloid P component from Pichia pastoris: production and characterization.

Human serum amyloid P component (SAP) was expressed in the methylotrophic yeast Pichia pastoris. SAP cDNA was placed under control of regulatory sequences derived from the alcohol oxidase gene (AOX1), and its protein product was secreted using the Saccharomyces cerevisiae alpha-mating factor signal sequence. Recombinant SAP (r-SAP) was produced in a bioreactor with computer controlled fed-batch mode and purified by use of a C-terminal histidine tag. The yield of purified r-SAP was 3-4mg from 1L supernatant and 5-6mg from 1L cell paste, indicating that the majority of the produced SAP was not secreted. Treatment of the cell paste with EDTA increased the yield further by about 30%. The N-terminal of r-SAP purified from the supernatant showed non-complete cleavage of the alpha-mating factor signal sequence. Purified r-SAP, analyzed under native conditions, was shown to be a decamer, like purified human SAP (h-SAP), with monomers of 27kDa. Each monomer had one N-glycosylation site, positioned at the same site as for h-SAP. r-SAP bound to antibodies produced against h-SAP. Furthermore, r-SAP bound to ds DNA and influenza A virus subunits in a Ca(2+)-dependent manner and inhibited influenza A virus hemagglutination. These results indicate that r-SAP produced in P. pastoris has the same biological activity as purified h-SAP.