Tissue gas tensions and tissue metabolites for detection of organ hypoperfusion and ischemia.

BACKGROUND: The aim of this study was to evaluate how tissue gas tensions and tissue metabolites measured in situ can detect hypoperfusion and differentiate between aerobic and anaerobic conditions during hemorrhagic shock. We hypothesized that tissue PCO(2) (PtCO(2)) would detect hypoperfusion also under aerobic conditions and detect anaerobic metabolism concomitantly with or earlier than other markers. METHODS: Prospective experimental animal study with eight anesthetized pigs subjected to a continuous blood loss ∼8% of total blood volume per hour until death. We measured cardiac index, organ blood flows, and tissue levels of PO(2), PCO(2), glucose, pyruvate, lactate, and glycerol in intestine, liver, kidney, and skeletal muscle. RESULTS: With reduction in blood flow to the organs under aerobic conditions, PtCO(2) increased ∼1-4 kPa from baseline. With the onset of tissue hypoxia there was a pronounced increase of PtCO(2), lactate, lactate-pyruvate (LP) ratio, and glycerol. Tissue pH and bicarbonate decreased significantly, indicating that metabolic acid was buffered by bicarbonate to generate CO(2). CONCLUSION: Moderate tissue hypoperfusion under aerobic conditions is associated with increased PtCO(2), in contrast to metabolic parameters of ischemia (lactate, LP ratio, and glycerol) which remain low. From the onset of ischemia there is a much more rapid and pronounced increase in PtCO(2), lactate, and LP ratio. PtCO(2) can be used as a marker of hypoperfusion under both aerobic and anaerobic conditions; it gives an earlier warning of hypoperfusion than metabolic markers and increases concomitantly with or earlier than other markers at the onset of tissue anaerobiosis.

Incorporation of alpha- and beta-LNA (locked nucleic acid) monomers in oligodeoxynucleotides with polarity reversals.

The thymidine monomers of LNA with both alpha- and beta-configuration are incorporated with polarity reversals (i.e., with 3'-3' and 5'-5' junctions) in oligodeoxynucleotides with beta- and alpha-configuration, respectively. A 5'-O-phosphoramidite of the beta-LNA monomer is synthesised. Large destabilisations of duplexes with both complementary DNA and RNA are observed for oligodeoxynucleotides containing the alpha-LNA monomer, whereas a duplex with complementary RNA of an alpha-oligodeoxynucleotide containing the beta-LNA monomer is not destabilised.

Synthesis of abasic locked nucleic acid and two seco-LNA derivatives and evaluation of their hybridization properties compared with their more flexible DNA counterparts.

To investigate the structural basis of the unique hybridization properties of LNA (locked nucleic acid) three novel LNA derivatives with modified carbohydrate parts were synthesized and evaluated with respect to duplex stabilities. The abasic LNA monomer (X(L), Figure 1) with the rigid carbohydrate moiety of LNA but no nucleobase attached showed no enhanced duplex stabilities compared to its more flexible abasic DNA counterpart (X, Figure 1). These results suggest that the exceptional hybridization properties of LNA primarily originate from improved intrastrand nucleobase stacking and not backbone preorganization. Two monocyclic seco-LNA derivatives, obtained by cleavage of the C1'-O4' bond of an LNA monomer or complete removal of the O4'-furanose oxygen atom (Z(L) and dZ(L), respectively, Figure 1), were compared to their acyclic DNA counterpart (Z, Figure 1). Even though they are more constrained than Z, the seco-LNA derivatives Z(L) and dZ(L) destabilize duplex formation even more than the flexible seco-DNA monomer Z.

The conformations of locked nucleic acids (LNA).

We have used 2D NMR spectroscopy to study the sugar conformations of oligonucleotides containing a conformationally restricted nucleotide (LNA) with a 2'-O, 4'-C-methylene bridge. We have investigated a modified 9-mer single stranded oligonucleotide as well as three 9- and 10-mer modified oligonucleotides hybridized to unmodified DNA. The single-stranded LNA contained three modifications whereas the duplexes contained one, three and four modifications, respectively. The LNA:DNA duplexes have normal Watson-Crick base-pairing with all the nucleotides in anti-conformation. By use of selective DQF-COSY spectra we determined the ratio between the N-type (C3'-endo) and S-type (C2'-endo) sugar conformations of the nucleotides. In contrast to the corresponding single-stranded DNA (ssDNA), we found that the sugar conformations of the single-stranded LNA oligonucleotide (ssLNA) cannot be described by a major S-type conformer of all the nucleotides. The nucleotides flanking an LNA nucleotide have sugar conformations with a significant population of the N-type conformer. Similarly, the sugar conformations of the nucleotides in the LNA:DNA duplexes flanking a modification were also shown to have significant contributions from the N-type conformation. In all cases, the sugar conformations of the nucleotides in the complementary DNA strand in the duplex remain in the S-type conformation. We found that the locked conformation of the LNA nucleotides both in ssLNA and in the duplexes organize the phosphate backbone in such a way as to introduce higher population of the N-type conformation. These conformational changes are associated with an improved stacking of the nucleobases. Based on the results reported herein, we propose that the exceptional stability of the LNA modified duplexes is caused by a quenching of concerted local backbone motions (preorganization) by the LNA nucleotides in ssLNA so as to decrease the entropy loss on duplex formation combined with a more efficient stacking of the nucleobases.