Phosphoinositide-dependent protein kinase-1 -LRB- PDK1 -RRB- - independent activation of the protein kinase C substrate , protein kinase D Phosphoinoisitide dependent kinase l -LRB- PDK1 -RRB- is proposed to phosphorylate a key threonine residue within the catalytic domain of the protein kinase C -LRB- PKC -RRB- superfamily that controls the stability and catalytic competence of these kinases . Hence , in PDK1-null embryonic stem cells intracellular levels of PKCα , PKCβ1 , PKCγ , and PKCε are strikingly reduced . Although PDK1-null cells have reduced endogenous PKC levels they are not completely devoid of PKCs and the integrity of downstream PKC effector pathways in the absence of PDK1 has not been determined . In the present report , the PDK1 requirement for controlling the phosphorylation and activity of a well characterised substrate for PKCs , the serine kinase protein kinase D , has been examined . The data show that in embryonic stem cells and thymocytes loss of PDK1 does not prevent PKC-mediated phosphorylation and activation of protein kinase D . These results reveal that loss of PDK1 does not functionally inactivate all PKC-mediated signal transduction . 1 Introduction Phosphoinoisitide dependent kinase l -LRB- PDK1 -RRB- phosphorylates a key threonine residue within the catalytic domain of a number of AGC family kinases , including diacylglycerol-regulated kinases of the protein kinase C -LRB- PKC -RRB- superfamily and phosphatidyl inositol-3 kinase -LRB- PI3K -RRB- - controlled serine kinases such as Akt/PKB and the 70-kilodalton ribosomal S6 kinase 1 -LRB- S6K1 -RRB- -LSB- 1 -RSB- . PDK1-mediated phosphorylation of many of these AGC kinases is essential for their activation . Accordingly , deletion of PDK1 by homologous recombination removes one of the rate-limiting regulators of multiple serine kinases and causes embryonic lethality -LSB- 2 -RSB- . To circumvent this prenatal death and explore the role of PDK1 in different tissues , mice expressing PDK1 alleles flanked with the loxP Cre excision sequence -LRB- PDK1flΔneo/flΔneo mice -RRB- have been generated . These were first used to achieve muscle specific deletion of PDK1 , producing mice that die early with severe defects in cardiac function -LSB- 3 -RSB- . The importance of PDK1 for T lymphocyte biology has also been explored by looking at T cell development following T lineage specific deletion of PDK1 . These studies showed that deletion of PDK1 in T cell progenitors in the thymus results in a block in T cell differentiation , indicating that PDK1 has essential functions in T lymphocyte development -LSB- 4 -RSB- . The importance of PDK1 for T cell development in the thymus focuses attention on the identification of the PDK1 substrates directly involved in mediating the unique and non-redundant functions of this kinase in vivo . In this context , studies of mouse embryonic stem cells in which both PDK1 alleles have been deleted have shown that PDK1 is necessary for T-loop phosphorylation , and catalytic activity , of the AGC kinases Akt/PKB , S6K1 and p90 ribosomal S6 kinase -LRB- p90RSK -RRB- -LSB- 5 -RSB- . However , PDK1 is not required for the phosphorylation and activation of other AGC kinases such as PKA or AMPK -LSB- 5 -RSB- . One very important subfamily of AGC kinases that can be regulated by PDK1 are the protein kinase C enzymes . There are multiple isoforms of PKC : the classical PKCs -LRB- α , βI , βII and γ -RRB- which are regulated by calcium , diacylglycerol -LRB- DAG -RRB- and phospholipids ; novel PKCs -LRB- δ , ε , η and θ -RRB- which are regulated by DAG and phospholipids ; and the atypical PKCs -LRB- ζ and λ -RRB- which are insensitive to both calcium and DAG . The catalytic domain of PKCs have a conserved T-loop motif that is a substrate for PDK1 , and it has been proposed that PDK1 is responsible for T-loop phosphorylation of the various PKC isoforms -LSB- 6 -- 8 -RSB- . PDK1-mediated T-loop phosphorylation of PKCs is proposed to stabilize protein expression by acting as a priming signal that enables PKCs to achieve catalytic maturity -LSB- 6,7 -RSB- . Here , PDK1 docks to a hydrophobic motif in the C-terminus of newly synthesised PKC enzymes , enabling it to phosphorylate their T-loop sites . Following T-loop phosphorylation , PDK1 disengages enabling PKCs to autophosphorylate their C-terminal hydrophobic motifs and achieve catalytic maturity -LSB- 9 -- 11 -RSB- . Once mature , PKCs remain inactive due to autoinhibition from their pseudosubstrate motifs and are only activated following the generation of second messengers , such as diacylglycerol and calcium . The release of PDK1 from PKCs is rate limiting and so PDK1 is responsible for controlling the amount of mature/catalytically competent PKCs expressed in cells -LSB- 11 -RSB- . Hence , in the absence of PDK1 , intracellular levels of PKCα , PKCβ1 , PKCγ , and PKCε are strikingly reduced -LSB- 6,12 -RSB- . PKCξ levels are similar in wild-type and PDK1-null embryonic stem cell -LRB- ES cells -RRB- although PKCξ is not phosphorylated on its T-loop Thr410 site in the absence of PDK1 -LSB- 6,13 -RSB- . PKCδ expression is also reduced in PDK1-null cells -LSB- 6,12 -RSB- although there was still phosphorylation of the PKCδ T-loop site Thr505 in the residual pool of PKCδ still present in PDK1-null cells -LSB- 13 -RSB- . Moreover , there is some evidence in the literature that T-loop phosphorylation is not absolutely critical for PKCδ catalytic activity , since a bacterially-expressed Thr505A PKCδ mutant protein retains some activity in vitro -LSB- 14 -RSB- . Of note , a glutamic acid residue at position 500 within the PKCδ activation loop , which is important for catalytic activity -LSB- 15 -RSB- , could potentially compensate for the lack of PKCδ T-loop phosphorylation in PDK1-null cells . These studies support the hypothesis that PDK1 has an important role in regulating PKC phosphorylation and stability . However , it has not been shown unequivocally that the reduced levels of PKCs in PDK1-null cells abrogate PKC-mediated cellular responses . In this context , it has been shown that in the absence of PDK1 there is loss of Akt/PKB and S6K1 T-loop phosphorylation and a corresponding inability of cells to regulate phosphorylation of Akt/PKB or S6K1 substrates -LSB- 5 -RSB- . There is thus confidence that Akt/PKB and S6K1 function is defective in PDK1-null cells . This has not been determined for the PKCs because although PDK1-null cells have reduced endogenous PKC levels the integrity of downstream PKC effector pathways in PDK1-null cells has not been determined . One well characterised target for novel PKCs is the serine kinase protein kinase D -LRB- PKD -RRB- . Activation of PKD requires the phosphorylation of two key serine residues within the catalytic domain -LSB- 16,17 -RSB- . These residues are absolutely conserved through evolution from C. elegans to man and are substrates for novel PKCs . The activation of PKCs is thus required for PKD activation in a variety of cell lineages in response to a number of different stimuli -LSB- 16,17 -RSB- . For example , it has been demonstrated that PKD activity in lymphocytes is regulated by PKC-mediated phosphorylation of its catalytic domain -LSB- 18,19 -RSB- and phosphorylation of the PKD catalytic domain is reduced in PKCβ-deficient spleenocytes -LSB- 20 -RSB- . It would therefore be expected that PKD would remain inactive if PKCs can not achieve catalytic maturity in PDK1-null cells . In support of this hypothesis , previous work has established that co-expression of PDK1 and the novel PKCε isoform , together with PKD , enhances PKD activity -LSB- 21 -RSB- . The hypothesis that PKD activation is dependent on PDK1 has not been fully investigated and is important to address as it is one way to assess whether there is residual functional PKC activity in PDK1-null cells . Accordingly , the objective of the present study was to examine whether loss of PDK1 results in loss of the PKC signalling pathway that regulates PKD . 2 Results and discussion 2.1 Activation of protein kinase D in PDK1 − / − thymocytes To investigate the impact of PDK1 loss on protein kinase D activation in PDK1 deficient T lymphocytes , mice expressing PDK1 alleles flanked with the loxP Cre excision sequence -LRB- PDK1flΔneo/flΔneo -RRB- were bred with transgenic mice expressing Cre recombinase under the control of the proximal p56lck promoter which induces expression of Cre in T cell progenitors in the thymus . It has been shown previously that in LckCre + PDK1flΔneo/flΔneo mice PDK1 is deleted in pre-T cells , preventing normal T cell development -LSB- 4 -RSB- . To explore the role of PDK1 in the regulation of PKD activity , wild-type or PDK1 − / − pre-T cells were isolated and left unstimulated , stimulated with a crosslinking α-CD3 antibody -LRB- to activate preTCR signalling -RRB- or were stimulated with the phorbol ester PdBu -LRB- a diacylglycerol mimetic which activates classical/novel PKCs -RRB- , as indicated . PKD catalytic activity was monitored using an antisera that recognises PKD molecules that are autophosphorylated on Ser916 -LSB- 22 -RSB- . The data -LRB- Fig. 1 -RRB- show that in quiescent wild-type thymocytes PKD is not autophosphorylated on Ser916 whereas α-CD3 or PdBu stimulation strongly induced PKD catalytic activity , as shown by increased Ser916 autophosphorylation . The results also show that both α-CD3 and PdBu treatment induce strong PKD Ser916 autophosphorylation in PDK1 − / − pre-T cells -LRB- Fig. 1 -RRB- . PKD activation is dependent on PKC-mediated phosphorylation of two key serine residues -LRB- Ser744/S748 -RRB- within the PKD catalytic domain and as shown in Fig. 1 , PKD is phosphorylated on Ser744/S748 following α-CD3 or PdBu activation in both wild-type and PDK1 − / − pre-T cells . The phosphorylation and activation of PKD in PDK1 − / − pre-T cells argues that these cells still contain a functional , catalytically competent PKC -LRB- s -RRB- that is able to respond to diacylglycerol/phorbol ester signals . RNAi experiments have demonstrated that PKCε in Swiss 3T3 cells or PKCδ in HeLa cells are the PKC isoforms responsible for serine phosphorylation -LRB- Ser744/S748 -RRB- within the PKD activation loop -LSB- 23,24 -RSB- . PKCδ has been shown to be residually phosphorylated on its T-loop Thr505 site in PDK1-null ES cells -LSB- 13 -RSB- . We therefore examined the phosphorylation status of the PKCδ T-loop site , Thr505 , in PDK1 − / − pre-T cells . The data in Fig. 1 show basal phosphorylation of PKCδ Thr505 in wild-type pre-T cells and also show that significant phosphorylation of PKCδ on Thr505 in retained in PDK1 − / − pre-T cells . In contrast , as previously demonstrated , PDK1 − / − pre-T cells have lost activity of the PKB/S6K1 signalling pathway that controls the phosphorylation of the ribosomal S6 subunit on Ser235/236 -LSB- 4,25 -RSB- . The data in Fig. 1 also explore the impact of PDK1 loss on the T-loop phosphorylation of p90RSK1 and PRK1 . The data show Western blot analysis of wild type and PDK1 null pre-T cells with a phospho-specific antibody against Ser227 in p90RSK1 and show that this site is basally phosphorylated in wild-type pre-T cells but is not phosphorylated in PDK1-null pre-T cells . Similarly , basal phosphorylation of PRK1 on its Thr774 T-loop site is substantially reduced in PDK1-null pre-T cells . Thus , PDK1-null pre-T cells show defective activation of some , but not all , AGC family kinases : regulation of a PKC -- PKD signalling pathway remains intact in these cells . 2.2 Activation of protein kinase D in PDK1 − / − ES cells The experiments in Fig. 1 show that PDK1 is not essential for PKD activation in thymocytes . To probe further the involvement of PDK1 in the regulation of PKC -- PKD signalling , experiments in wild-type and PDK1 − / − mouse embryonic stem cells were performed . Western blot analysis of wild-type and PDK1 − / − ES cells with a PKCδ Thr505 phosphospecific antisera confirm that PKCδ Thr505 phosphorylation is detectable , albeit slightly reduced , in PDK1-null versus wild-type ES cells -LRB- Fig. 2A -- C -RRB- . To probe PKD activity , cells were left unstimulated or activated by exposure to serum or phorbol ester as indicated and PKD catalytic activity monitored using the pSer916 antisera . In quiescent , wild-type ES cells , PKD was not autophosphorylated on Ser916 but both serum and PdBu stimulation strongly and rapidly induced PKD activity as measured by increased Ser916 autophosphorylation -LRB- Fig. 2A and B -RRB- . Serum or PdBu stimulation also induced PKD activity in the PDK1 − / − ES cells -LRB- Fig. 2A and B -RRB- whereas p90RSK1 T-loop phosphorylation -LRB- Ser227 -RRB- was basally detectable in wild-type ES cells but not in the PDK1 − / − ES cells -LRB- Fig. 3 -RRB- . S6K1 signalling was also abrogated , as expected , in the PDK1 − / − ES cells , as shown by the lack of phosphorylation of its substrate , the S6 ribosomal protein subunit -LRB- Fig. 2B -RRB- . The ability of serum and phorbol esters to activate PKD in PDK1-null ES cells is an indication that there is residual , catalytically competent PKC in these cells . PKC-mediated activation of PKD can be prevented by the PKC inhibitor GF109203X -LSB- 17 -RSB- . Therefore , the effects of GF109203X , on PKD activation in wild-type or PDK1 − / − ES cells was examined . The data -LRB- Fig. 3 -RRB- show that PKD was phosphorylated on Ser916 in activated PDK1 + / + and PDK1 − / − cells but that PKD autophosphorylation on Ser916 was prevented by prior treatment of both cell types with GF109203X . 3 Concluding remarks Loss of PDK1 in ES cells causes a reduction in levels of PKCα , βI , γ , δ , and ε -LSB- 6,12 -RSB- but it was not resolved whether the residual PKC in these cells is functional . As discussed above , there is a large body of evidence that PKD activation is mediated by novel PKCs such as PKCδ and ε and it has previously been proposed that PDK1 is required for activation of PKD -LSB- 21 -RSB- , reflecting the requirement of PDK1 for phosphorylation of the key priming T-loop phosphorylation sites in PKCs . However , the present study shows that the hypothesis that PKD activation is dependent on PDK1 is incorrect . The reason this model fails is that there is not a complete loss of PKC activity in the absence of PDK1 . We show that in PDK1-null pre-T cells PKCδ is still phosphorylated on its T-loop site Thr505 ; hence activation of pre-T cells with phorbol esters can still induce phosphorylation of PKD on its PKC substrate sequences Ser744/S748 , inducing PKD activation and subsequent autophosphorylation on Ser916 . In addition , the present data show that PKD can also be activated by phorbol esters or serum stimulation of PDK1-null ES cells in a PKC-regulated manner . Thus , the present results showing activation of PKD in PDK1 − / − cells is evidence that at least one PKC-mediated signalling pathway is intact in the absence of PDK1 . While loss of PDK1 does not prevent the activation of PKD , the issue of whether the efficiency of PKD activation is affected in PDK1-null cells requires further experimentation . While robust activation of PKD signalling is observed in PDK1-null ES cells and pre-T cells in response to phorbol esters or CD3 crosslinking we can not exclude the possibility that PKD activation may be compromised in PDK1-null mature peripheral T cells during physiological peptide : MHC stimulation , however these experiments await the development of genetic models to test this . In summary , PDK1 is essential for pre-T cell development and its loss is associated with loss of PKB/S6K1-mediated signal transduction -LSB- 4,25 -RSB- . The present results now show loss of p90RSK and PRK1 T-loop phosphorylation in PDK1 − / − pre-T cells but reveal that the PKCδ T-loop site Thr505 is still phosphorylated in PDK1-null pre-T cells . It has been proposed that the Thr505 T-loop site in PKCδ -LRB- as well as the corresponding T-loop site in PKCθ , -LSB- 26 -RSB- -RRB- can be autophosphorylated , which may explain why PKCδ Thr505 is still phosphorylated in the PDK1-null cells . Alternatively the present results could argue for the existence of another PKCδ priming kinase in developing thymocytes . Whatever the molecular basis for the continued phosphorylation of PKCδ following the loss of PDK1 , the present results show that there is still residual functional PKC activity once PDK1 is lost . Hence , the importance of PDK1 for T cell development must reflect the requirement for PDK1 mediated phosphorylation of other members of the AGC serine kinase family , such as Akt/PKB , S6K1 or p90RSK , since phosphorylation and activity of these kinases is strictly dependent on PDK1 expression . 4 Materials and methods 4.1 Cell preparation and stimulation PDK1 + / + and PDK1 − / − murine embryonic stem cells -LSB- 5 -RSB- were grown on gelatinised tissue culture plates in Knockout DMEM supplemented with 10 % knockout serum replacement , 15 % foetal bovine serum -LRB- Hyclone -RRB- , 0.1 mM non-essential amino acids , antibiotics -LRB- 100 U/ml penicillin G , 100 μg/ml streptomycin and 1 μg/ml ciprofloxacin -LSB- Bayer Pharmaceuticals -RSB- -RRB- , 2 mM l-glutamine , 1 mM sodium pyruvate , 0.1 mM β-mercaptoethanol and 25 ng/ml murine leukaemia inhibitory factor -LRB- LIF -RRB- . 4.2 Mice All mice used were between 5 and 7 weeks of age . Mice were bred and maintained in specific pathogen-free conditions and animal experimentation was approved by Home Office project license PPL60/3116 . T-PDK1 − / − mice were generated by crossing mice with PDK1 floxed alleles -LRB- PDK1flΔneo/flΔneo -RRB- with mice expressing Cre recombinase under the control of the p56lck promoter to ablate expression of PDK1 in T cells -LSB- 2,4 -RSB- . Control mice used for analyses of T-PDK1 − / − mice were age-matched phenotypically normal littermates . 4.3 Purification of thymocyte subpopulations Antibodies conjugated to fluorescein isothiocyanate -LRB- FITC -RRB- , phycoerythrin -LRB- PE -RRB- , allophycocyanin -LRB- APC -RRB- and biotin were obtained from PharMingen . Cells were stained for surface expression of the following markers : CD4 , CD8 , CD25 , CD44 , CD3ε , γδ , B220 , and Thy1 . The DN4 subpopulation were defined by their cell surface marker expression and subsequently sorted using a Vantage cell sorter -LRB- Becton Dickinson -RRB- or AutoMacs magnetic cell sorter -LRB- Miltenyi Biotec -RRB- . 4.4 Cell lysis and Western blot analysis Cells were lysed for 10 min at 4 °C -LRB- 20 × 106 cells/ml -RRB- using buffer : 100 mM HEPES , pH 7.4 , 150 mM NaCl , 1 % NP40 , 20 mM sodium fluoride , 20 mM iodoacetamide , 2 μM EDTA , 1 mM sodium orthovanadate , 2 μg/ml pepstatin A , 2 μg/ml leupeptin , 2 μg/ml chymostatin , 2 μg/ml antipain , 40 mM β-glycerophosphate and 1 mM phenylmethylsulfonylflouride . Soluble proteins were concentrated by precipitation with 1.5 vol . of acetone and separated by 8 % sodium dodecyl sulfate -- polyacrylamide gel electrophoresis -LRB- SDS -- PAGE -RRB- , transferred to polyvinylidene difluoride membranes and detected by Western blot analysis with the indicated antibodies -LRB- Cell Signalling Technology -RRB- .

O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O 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B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O B O O O O O B O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O B O O O O O O B O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O B O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

pdk1, 3′-phosphoinositide-dependent protein kinase-1

s6k1, 70-kilodalton ribosomal s6 kinase 1


Assessment of stroke volumeindex with three different bioimpedance algorithms : lack of agreement compared to thermodilution Objective The accuracy of bioimpedance stroke volumeindex -LRB- SVI -RRB- is questionable as studies report inconsistent results . It remains unclear whether the algorithms alone are responsible for these findings . We analyzed the raw impedance data with three algorithms and compared bioimpedance SVI to transpulmonary thermodilution -LRB- SVITD -RRB- . Introduction Additional information about the cardiovascular status of critically ill patients can be obtained by measuring cardiac output -LRB- CO -RRB- . Pulmonary artery thermodilution CO monitoring has remained the reference technique for three decades -LSB- 1 -RSB- but is invasive and associated with specific complications -LSB- 2 -- 4 -RSB- . Thoracic bioimpedance cardiography , a noninvasive CO monitoring technique , exhibits many qualities of the ideal CO monitor : it is operator independent , continuous , and cost-effective -LSB- 5 -RSB- . Since the late 1960s a number of bioimpedance devices have been developed with cylinder - or cone-based models of a homogeneously with blood filled human thorax . Method comparison studies have demonstrated conflicting results with respect to validity and reliability -LSB- 6 -RSB- , varying from satisfactory correlations -LSB- 7 -- 9 -RSB- to poor correlations -LSB- 10 , 11 -RSB- . Inaccuracies can result from irregular cardiac rhythms , abnormal ventilatory patterns , motion artifacts , valvular heart diseases , electrocautery , changes in hematocrit , excessive changes in body temperature , and an obese body habitus -LSB- 5 -RSB- . Thereby , it remains unclear whether the methodology -LRB- i.e. detection of impedance signals from the thorax using a small number of electrodes -RRB- per se or limitations of the underlying algorithms are responsible for these conflicting results . We hypothesized that bioimpedance SV measured with any of three well-established bioimpedance algorithms is valid and reliable . We compared bioimpedance stroke volume index -LRB- SVI -RRB- with transpulmonary thermodilution stroke volume index -LRB- SVITD -RRB- as a reference of proven accuracy -LSB- 12 -RSB- . Materials and methods After approval by the institutional review board and written informed consent , patients scheduled for elective coronary artery bypass graft -LRB- CABG -RRB- surgery with cardiopulmonary bypass were included . Exclusion criteria were : ejection fraction less than 40 % , femoral arterialdisease , and valvular heart disease . A total intravenous anesthesia technique was used during the operation . Normocapnia was maintained during mechanical ventilation -LRB- inspired fraction of oxygen 0.4 , positive end-expiratory pressure 5 cmH2O -RRB- . A4-Fthermodilutioncatheter -LRB- PulsiocathPV2014 L16 -RRB- was introduced into the femoral artery and connected to a commercially available CO device -LRB- PiCCO , Pulsion , Munich , Germany -RRB- . Transpulmonary thermodilution cardiac output -LRB- TPCO -RRB- was measured by quadruple injections of 15 ml ice-cold saline into the right atrium and used for transpulmonary thermodilution stroke volume calculation . After rubbing and cleaning the skin with alcohol to achieve a skin-to-electrode impedance as low as possible , two `` current injecting '' electrodes were placed on the forehead and the left hip , and two voltage sensing electrodes were placed on the lateral side of the neck just above the left clavicle and in the left midaxillary line at the level of the sternal xiphoid . An alternating current of 0.3 mA -LRB- 64 kHz -RRB- was applied . A thoracic bioimpedance cardiograph -LRB- HL-4 , Hemologic , Amersfoort , The Netherlands -RRB- was used for recording raw bioimpedance signals in the perioperative period . The first derivative of the thoracic impedance -LRB- dZ/dt -RRB- and the electrocardiographic signal were displayed on the screen . Raw data were analyzed off-line over a 20-s period -LRB- LabView , E-solutions , Arnhem , The Netherlands -RRB- and used for bioimpedance SV calculation using three distinct reconstruction algorithms : Kubicek et al. -LSB- 13 -RSB- , Sramek -- Bernstein -LSB- 14 -RSB- , and Wang et al. -LSB- 15 -RSB- . Data collected after induction before skin incision -LRB- t1 -RRB- , after weaning from cardiopulmonary bypass -LRB- t2 -RRB- , after sternal closure -LRB- t3 -RRB- , after admission at the intensive care -LRB- t4 -RRB- , after reaching normothermia -LRB- 36.5 °C -RRB- -LRB- t5 -RRB- , after extubation -LRB- t6 -RRB- , and before discharge to the ward -LRB- t7 -RRB- were : heart rate , mean arterial pressure , central venous pressure , bioimpedance raw data and TPCO measurements . SVI was calculated by dividing stroke volume by body surface area . Sample size calculation was performed to limit the width of a 95 % confidence interval for the mean error ; based on a mean CO of 5.0 l/min , a correlation coefficient of 0.65 , a mean error of 30 % -LSB- 16 -RSB- , and a confidence interval of 95 % , a sample size of 20 patients was calculated . Statistical analysis was performed using PRISM 4.0 -LRB- GraphPad , San Diego , Calif. , USA -RRB- and SPSS 12.0.2 -LRB- SPSS , Chicago , Ill. , USA -RRB- . If the analysis of variance revealed a significant interaction , post-hoc analysis was performed using Student 's t test with Bonferroni 's correction . Validity and reproducibility between bioimpedance SVI and SVITD were tested according to Bland and Altman -LSB- 17 -RSB- : bias , precision -LRB- = SD of bias -RRB- , limits of agreement -LRB- LOA -RRB- , and mean error -LSB- 15 -RSB- for absolute SVI values and for relative changes in SVI -LRB- ΔSVI -RRB- . Mean error was calculated as 2 × precision divided by the mean SVITD . Pooled data and data per time moment were analyzed . A p value less than 0.05 was considered to indicate statistical significance . Results The population studied included 15 men and 5 women -LRB- age 64 ± 10 years , weight 79 ± 12 kg , height 171 ± 8 cm ; body surface area 1.64 -- 2.20 m2 -RRB- . Of 140 SVI series obtained 93 with each technique were available for statistical analysis . Forty seven series of SVI data could not be used for further analysis because of failure to obtain SVI due to insufficient raw bioimpedance signals . Time course of SVI and Bland -- Altman analysis for each method are shown in Fig . 1 . Bias , precision , LOA , and mean error between SVITD and SVIK were 1.0 ± 10.8 ml/m2 , − 20.2 to +22.1 ml/m2 , and 63 % , respectively , while the results for SVITD and SVISB were 9.8 ± 11.4 ml/m2 , -- 12.5 to +32.2 ml/m2 , and 67 % and for SVITD and SVIW − 15.7 ± 8.2 ml/m2 , − 31.6 to +0.3 ml/m2 , and 48 , % respectively . Analysis of bioimpedance data for each algorithm at each time point are given in Table 1 . Fig. 1Time course of stroke volume index -LRB- SVI -RRB- and Bland -- Altman analysis obtained by each method . SVITD , Transpulmonary thermodilution stroke volume index ; SVIK , stroke volume index according to Kubicek et al. -LSB- 13 -RSB- ; SVISB , stroke volume index according to Sramek -- Bernstein -LSB- 14 -RSB- ; SVIW , stroke volume index according to Wang et al. -LSB- 15 -RSB- . * p < 0.05 vs. t1Table 1Bias , precision , limits of agreement -LRB- LOA -RRB- , and mean error of stroke volume index calculated with three bioimpedance algorithms vs. transpulmonary thermodilution stroke volume index -LRB- SVITD -RRB- for each time moment -LRB- t1 , after induction before skin incision ; t2 , after weaning from CPB ; t3 , after sternal closure ; t4 , after admission at the intensive care unit ; t5 , after reaching normothermia at 36.5 °C ; t6 , after extubation ; t7 , before discharge to the ward ; SVIK , stroke volume index according to Kubicek et al. -LSB- 13 -RSB- ; SVISB , stroke volume index according to Sramek -- Bernstein -LSB- 14 -RSB- ; SVIW , stroke volume index according to Wang et al. -LSB- 15 -RSB- -RRB- SVIKSVISBSVIWtime Bias -LRB- ml/m -- 2 -RRB- Precision -LRB- ml/m -- 2 -RRB- LOA -LRB- ml/m -- 2 -RRB- Mean error -LRB- % -RRB- Bias -LRB- ml/m -- 2 -RRB- Precision -LRB- ml/m -- 2 -RRB- LOA -LRB- ml/m -- 2 -RRB- Mean error -LRB- % -RRB- Bias -LRB- ml/m -- 2 -RRB- Precision -LRB- ml/m -- 2 -RRB- LOA -LRB- ml/m -- 2 -RRB- Mean error -LRB- % -RRB- t16 .413.9 -- 20.8 , +33.67715.512.3 -- 8.6 , +39.668 -- 14.79.8 -- 33.9 , +4.554 t20 .68.8 -- 16.6 , +17.85111.89.3 -- 6.4 , +30.054 -- 13.04.8 -- 22.5 , -- 3.528 t3 -- 3.07.8 -- 18.3 , +12.4516.213.9 -- 21.0 , +33.490 -- 14.28.1 -- 30.2 , +1.753 t41 .08.4 -- 15.5 , +17.6569.95.4 -- 0.7 , +20.436 -- 13.85.8 -- 25.1 , -- 2.538 t50 .410.0 -- 19.2 , +20.05810.110.7 -- 10.9 , +31.162 -- 16.38.6 -- 33.1 , +0.650 t61 .513.3 -- 24.5 , +27.5737.212.1 -- 16.4 , +30.966 -- 17.89.5 -- 36.4 , +0.852 t7 -- 0.610.7 -- 21.5 , +20.4608.313.0 -- 17.2 , +33.872 -- 18.08.1 -- 34.0 , -- 2.145 Discussion This study compared three bioimpedance algorithms assessing bioimpedance SVI to SVITD during the perioperative period in CABG patients . However , significant deviations were found , and accurate clinical decision making was not possible based on absolute values or changes in bioimpedance SVI . No single algorithm was superior to another . Interestingly , application of the Wang algorithm produced consistent underestimation , whereas the two other algorithms overestimated SVI . Our study differed from previous studies in several important aspects . Raw voltage data were measured and used for off-line calculation of bioimpedance SVI on the basis of different bioimpedance algorithms commonly used in commercially available devices . Therefore data were obtained without using different bioimpedance devices and calculation was independent from built-in proprietary software algorithms . Measurements were performed not only in the operating room but also in the intensive care in ventilated as well as in spontaneously breathing patients . The difference between bioimpedance SVI and SVITD for any of the three algorithms may be explained by the fact that the relationship between the signal on the voltage sensing electrodes and the resulting SVI is based on assumptions in relation to multiple effects . Whereas SV is equal to the change in the left ventricular volume during the systole , the voltage signal measured with bioimpedance is a result of volume changes in different intrathoracic compartments during the cardiac cycle , such as the intracardiac cavities , aorta , superior and inferior vena cava , and pulmonary circulation on the `` injected '' current -LSB- 18 -RSB- . Vascular diseases -LRB- atherosclerosis -RRB- can affect the relative contribution of the aorta to the bioimpedance signal because the volume change in the aorta during the cardiac cycle depends on aortic compliance . Moreover , a considerable anatomical variability exists between patients and within the cardiac cycle . The orientation of the central heart axis in relation to the thorax cavity varies considerably between patients but also during the cardiac cycle . Both influence the main current density field and hence the relative contribution of SV to the bioimpedance signal . It is questionable whether it is even possible to measure SVI reliably using thoracic bioimpedance with only one single voltage input stream given the fact that each of three distinctly different algorithms failed to produce satisfactory agreement with SVITD . Therefore an increase in the number of data input streams -LRB- i.e. electrodes -RRB- may improve the validity and reliability of the technique . Consequently suggestions have been made to optimize the measurement technique and the basic bioimpedance SV equation -LSB- 19 -RSB- . Recently Spiess et al. -LSB- 8 -RSB- and Sageman et al. -LSB- 9 -RSB- studied a second-generation thoracic bioimpedance cardiograph -LRB- BioZ System 1.52 , Cardiodynamics International , San Diego , Calif. , USA -RRB- in CABG patients and found a clinically acceptable correlation between pulmonary artery thermodilution and bioimpedance . However , mean error in the study by Spiess et al. was 26 % after induction of anesthesia and exceeded the clinically acceptable 30 % during the other measurements -LSB- 8 -RSB- . In contrast , our study showed a mean error exceeding 30 % with the exception of t2 using the Wang algorithm . In conclusion , common cylinder - and cone-based models for bioimpedance SVI calculation are not reliable compared to SVITD measurements in CABG patients . These models are oversimplifications of the complex electrical events occurring inside the thorax during the cardiac cycle . The problem of retrieving SV from voltage data may be considered as a special case of the general inverse conductivity theory -LSB- 20 -RSB- . There is need for a more robust mathematical approach -LRB- see Electronic Supplementary Material -RRB- , including an increase in the number of voltage measurement input streams , an accurate description of the physics of current density distributions and taking into account the full spectrum of all relevant patient anatomical variabilities . Electronic supplementary material Electronic Supplementary Material -LRB- DOC 54K -RRB-

O O B I O O O B O O O O O O O O O O O O B B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B I O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O B O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I B I O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

method comparison
cardiac output
stroke volumeindex
bioimpedance
transpulmonary thermodilution
coronary artery bypass graft