dogs : four adult male and 4 adult female beagle dogs were used in this 
 study .
it was carried out in accordance with hokkaido university guidelines for the care and 
 use of experimental animals , which basically conform to those of the association for 
 assessment and accreditation of laboratory animal care ( aaalac ) international .
the study was 
 approved by the committee for laboratory animals , graduate school of veterinary medicine , 
 hokkaido university ( approval number 08 - 0473 ) . the ultracentrifugation - precipitation method for canine lipoprotein 
 analysis : the ultracentrifugation - precipitation ( u - p ) method for canine 
 lipoprotein analysis
briefly , blood samples 
 were collected from dogs that had been fasted for 24 hr .
peripheral blood was taken into 
 tubes with edta , and the plasma was separated by centrifugation at 1,500  
 g for 10 min at 4c .
four milliliters of the plasma was placed in a 
 thermoplastic ultracentrifuge tube and overlayered with 6 ml of 154 mm 
 nacl . the tube was capped and centrifuged at 164,000  g for 18 hr at 4c 
 in an ultracentrifuge ( beckman l7 - 55 ultracentrifuge , beckman coulter inc . , brea , ca , 
 u.s.a . ) .
three milliliters of the upper layer , which included lipoproteins with a density of 
 less than 1.006 g / ml(vldl ) , were removed as the vldl fraction .
seven 
 milliliters of the lower layer containing lipoproteins with a density greater than 1.006 
 g / ml(hdl and ldl ) were collected as the hdl+ldl fraction .
the ldl in the 
 hdl+ldl fraction was precipitated by incubation with 400 l of 1 
 u/l heparin for 10 min and 5 ml of 101 mm 
 mncl2 for 30 min on ice .
after centrifugation at 10,800  g 
 for 30 min at 4c , the resultant supernatant was collected as the hdl fraction , and the 
 sediment was collected as the ldl fraction .
measurement of cholesterol concentration in fractions separated by the 
 ultracentrifugation - precipitation method : the cholesterol concentrations of 
 intact plasma ( total cholesterol ) , the hdl+ldl fraction and the hdl fraction 
 ( hdl - cholesterol , hdl - c ) were measured using an enzymatic assay ( chorestest , 
 sekisui medical co. , ltd . ,
tokyo , japan ) with a spectrophotometer ( u-3210 , hitachi , tokyo , 
 japan ) .
the ldl - cholesterol ( ldl - c ) concentration was calculated as the difference between 
 the cholesterol concentration of the hdl+ldl fraction and that of the hdl fraction .
for the 
 u - p method , the ldl / hdl ratio was calculated from the ldl - c and hdl - c concentrations 
 calculated above .
analysis of canine lipoprotein using gp - hplc : lipoprotein analyses of 
 intact plasma and the hdl+ldl and hdl fractions were performed using commercial gp - hplc 
 service ( lipotest , spectrum lab .
the concentrations of hdl - c 
 and ldl - c determined by the u - p method were compared with those determined by gp - hplc .
lc - ms / ms sample preparation by tryptic digestion : lc - ms / ms sample 
 preparation was performed using the methods of rosenfeld et al .   and wilm et al .   with some modifications .
briefly , 7 microliters of 
 the hdl+ldl , hdl and ldl fractions , separated using the u - p method , were subjected to sds 
 polyacrylamide gel electrophoresis ( sds - page ) with 420% gradient acrylamide gels ( ready gel 
 j , bio - rad laboratories , inc . ,
the stained protein s bands were excised 
 with a scalpel , washed with 100 l of 50% acetonitrile in 25 mm ammonium 
 carbonate and left to dry at rt for 10 min with shaking in an eppendorf thermomixer .
the gel 
 slices were dehydrated with 100 l acetonitrile and dried in a vacuum 
 centrifugal dryer ( thermo savant spd speedvac , with a savant uvs 400 universal vacuum 
 system , thermo fisher scientific inc . ,
then , the gel slices were 
 alkylated with 55 mm iodoacetamide and 25 mm ammonium carbonate in light interception with 
 aluminum foil for 45 min .
, madison , wi , u.s.a . ) was added to the gel slices , and the gel 
 slices were added to 50 mm ammonium carbonate and incubated on ice for 30 min .
after 
 absorption of the protease solution , the gel slices were incubated at 37c for 12 hr .
to 
 extract hydrophilic and hydrophobic peptides from the gel slices , we performed a three - step 
 extraction . the first extracted solution ( 50% acetonitrile and 5% trifluoroacetic acid 
 [ tfa ] ) was added to the gel slices and then incubated in an ultrasonic bath ( usc-6 , osada , 
 inc .
next , the second extracted solution ( 99% 
 acetonitrile and 0.1% tfa ) was added to the gel slices and incubated in an ultrasonic bath 
 for 10 min .
finally , the third extraction ( 99.9% ultrapure water and 0.1% tfa ) was added to 
 the gel slices and incubated in an ultrasonic bath .
these samples produced by the three - step 
 extraction were concentrated to ~20 l in the vacuum centrifugal dryer .
lc - ms / ms analysis : the samples prepared above were analyzed by fourier 
 transform mass spectrometry ( ltq orbitrap , thermo fisher scientific inc .
, waltham , ma , 
 u.s.a . ) interfaced with the ultimate 3000 nano lc systems ( thermo fisher scientific inc . ) 
 equipped with a trap column ( zorbax 300 sb - c18 , 0.3  5 mm , agilent technology , santa clara , 
 ca , u.s.a . ) and analytical column ( acclaim pepmap100 3 m 100  , dionex 
 corp . ,
sunnyvale , ca , u.s.a . ) in acetonitrile gradient ( acetonitrile : water=70:30 , v / v ) .
peak 
 lists were generated from raw lc - ms / ms spectra using xcalibur 2.0.7 ( thermo fisher 
 scientific inc . ) .
the raw data acquired by the mass spectrometry experiment were searched 
 against the national center for biological information ( ncbi ) databases 
 ( www.ncbi.nlm.nih.gov ) using proteome discoverer 1.0 ( thermo fisher scientific inc . ) . statistical analysis :
the correlation of the hdl - c and ldl - c 
 concentrations between the u - p method and gp - hplc was investigated by pearson s correlation 
 coefficient test .
results were considered as correlated when r was significantly different 
 from 0 ( p<0.05 ) and over 0.7 .
analysis of canine lipoproteins in fractions separated using the 
 ultracentrifugation - precipitation method : first , intact canine plasma , the hdl 
 plus ldl fractions and hdl fractions separated by the u - p method were analyzed using 
 gp - hplc .
the peaks of vldl , ldl and hdl cholesterol were identified in intact plasma , but no 
 peak was observed for cm cholesterol , because of the 24 hr fast ( fig .
1.gp-hplc of canine lipoprotein in intact dog plasma ( a ) and of the hdl+ldl fraction 
 ( b ) and hdl fraction ( c ) separated by the u - p method .
the hdl+ldl fraction showed peaks for ldl and hdl cholesterol , but not for vldl 
 cholesterol ( fig .
1c ) . 
 next , the concentrations of hdl and ldl cholesterol determined using the u - p method were 
 compared with those determined by gp - hplc .
the canine hdl - c concentration determined by the 
 u - p method correlated well ( r=0.94 ) , but was not the same as that determined by gp - hplc 
 ( fig .
2.correlation of hdl - c ( a ) and ldl - c ( b ) concentrations and ldl / hdl ratio ( c ) between 
 the u - p method and gp - hplc .
the concentrations of canine hdl - c determined by the u - p method were lower than 
 those determined by gp - hplc .
the canine ldl - c concentration determined by the u - p method 
 correlated well with that determined by gp - hplc ( r=0.93 ) ( fig .
however , the concentrations of canine ldl - c determined by the u - p method 
 were higher than those determined by gp - hplc . the canine ldl / hdl ratio determined by the u - p 
 method correlated with that determined by gp - hplc ( r=0.75 ) , but was not the same as that 
 determined by gp - hplc ( fig . 2c ) .
gp - hplc of canine lipoprotein in intact dog plasma ( a ) and of the hdl+ldl fraction 
 ( b ) and hdl fraction ( c ) separated by the u - p method .
correlation of hdl - c ( a ) and ldl - c ( b ) concentrations and ldl / hdl ratio ( c ) between 
 the u - p method and gp - hplc .
sds - page of the canine hdl+ldl , hdl and ldl fractions : the canine hdl+ldl , 
 hdl and ldl fractions separated by the u - p method were subjected to sds - page .
it was found 
 that the canine hdl+ldl , hdl and ldl fractions from 3 healthy dogs included several 
 proteins , and the amounts of these proteins were different in each dog ( fig . 3fig .
3.sds-page analysis of the hdl+ldl ( a ) , hdl ( b ) and ldl ( c ) fractions .
the hdl+ldl , hdl 
 and ldl fractions separated from plasma of three healthy dogs by the u - p method were 
 analyzed using sds - page with the gradient gels .
lanes 1 , 2 and 3 , respectively , 
 indicate dogs 1 , 2 and 3 .
proteins with estimated molecular weights of approximately 
 25 ( a ) , 35 ( b ) and 60 kda ( c ) were detected in both the hdl and the ldl fractions . the 
 protein with a molecular weight of more than 250 kda ( d )
proteins with an estimated molecular weight of 
 greater than 250 kda and of approximately 25 kda were detected in all dogs ( fig .
, proteins with an 
 estimated molecular weight of approximately 25 kda were detected in all dogs ( fig .
proteins 
 with an estimated molecular weight of approximately 35 and 60 kda were also detected ( fig .
sds - page analysis of the hdl+ldl ( a ) , hdl ( b ) and ldl ( c ) fractions .
the hdl+ldl , hdl 
 and ldl fractions separated from plasma of three healthy dogs by the u - p method were 
 analyzed using sds - page with the gradient gels .
lanes 1 , 2 and 3 , respectively , 
 indicate dogs 1 , 2 and 3 .
proteins with estimated molecular weights of approximately 
 25 ( a ) , 35 ( b ) and 60 kda ( c ) were detected in both the hdl and the ldl fractions .
the 
 protein with a molecular weight of more than 250 kda ( d ) was detected only in the ldl 
 fraction .
lc - ms / ms analysis of the canine apolipoprotein a - i and apolipoprotein 
 b-100 : the proteins with a molecular weight of 25 kda in the hdl fraction were 
 analyzed using lc - ms / ms .
( a ) sequence coverage 
 ( coverage ) , molecular weight ( mw [ da ] ) and description of the analyzed protein are 
 shown as a table .
accession # , ncbi accession number ; # peptides , number of peptides ; 
 # aas , number of amino acids .
( b ) the coincident amino acid sequence between the 
 analyzed protein and the canine apo a - i ( gray highlighted sequence ) . the proteins were identified significantly as apo a - i ( fig .
4 ) . the proteins with a molecular weight of more than 250 kda in 
 the ldl fraction
were also analyzed using the lc - ms / ms and identified significantly as 
 canine apo b-100 ( fig . 5fig .
( a ) sequence coverage 
 ( coverage ) , molecular weight ( mw [ da ] ) and description of the analyzed protein are 
 shown as a table .
accession # , ncbi accession number ; # peptides , number of peptides ; 
 # aas , number of amino acids .
( b ) the coincident amino acid sequence between the 
 analyzed protein and the apo b-100 ( gray highlighted sequence ) . ) .
the proteins with a molecular weight of 60 kda were identified as canine albumin 
 ( data not shown ) .
the proteins with a molecular weight of 35 kda were identified as 
 haptoglobin ( data not shown ) .
( a ) sequence coverage 
 ( coverage ) , molecular weight ( mw [ da ] ) and description of the analyzed protein are 
 shown as a table .
accession # , ncbi accession number ; # peptides , number of peptides ; 
 # aas , number of amino acids .
( b ) the coincident amino acid sequence between the 
 analyzed protein and the canine apo a - i ( gray highlighted sequence ) .
( a ) sequence coverage 
 ( coverage ) , molecular weight ( mw [ da ] ) and description of the analyzed protein are 
 shown as a table .
accession # , ncbi accession number ; # peptides , number of peptides ; 
 # aas , number of amino acids .
( b ) the coincident amino acid sequence between the 
 analyzed protein and the apo b-100 ( gray highlighted sequence ) .
the present study examined the u - p method for its suitability as a gold standard method for 
 canine lipoprotein analysis .
since it was reported that gp - hplc was an efficient method in 
 small animals , the u - p method was compared with 
 gp - hplc first . in the u - p method
, an ultracentrifuge separates vldl particles by specific 
 gravity , and a heparin manganese chloride solution precipitates ldl particles .
gp - hplc separates the particles based on size by using 
 a specialized column .
it was confirmed that vldl 
 particles were removed from canine plasma by ultracentrifugation and that ldl was removed 
 from the hdl+ldl fraction by precipitation .
moreover , the hdl - c and ldl - c concentrations 
 determined by the u - p method correlated well with those determined by gp - hplc , although the 
 results of the u - p method were not consistent with those of gp - hplc .
it was suggested that 
 some of the canine hdl could be precipitated with heparin manganese chloride solution , 
 resulting in a higher ldl - c concentration and lower hdl - c concentration in the u - p method 
 than in gp - hplc .
furthermore , the presence in canine plasma of an apolipoprotein e ( apo 
 e)-rich subfraction of hdl with a similar density to ldl and apo e , which may co - precipitate 
 with ldl under certain conditions , may invalidate some of the methodologies used for the 
 measurement of canine ldl - c and hdl - c concentrations .
therefore , the hdl and ldl fractions separated by the u - p method were analyzed 
 further by lc - ms / ms . in the present study , analysis of the enzymatic fragment by lc - ms
/ ms was obviously an 
 important way of detecting canine apo a - i and apo b-100 . in the hdl fraction ,
this result shows that the hdl fraction contained only lipoprotein having apo a - i . 
 in the ldl fraction ,
this result shows that the 
 ldl fraction contains lipoproteins having apo a - i and apo b-100 .
it 
 is possible that a portion of the canine hdl was precipitated with the heparin manganese 
 chloride solution .
the canine hdl that was precipitated with canine ldl might include apo e , 
 as barrie et al . suggested .
it is 
 known that an apo e - rich subfraction of hdl is present in canine plasma   and has a similar density and behavior to ldl , although this indication is not studied .
accordingly , 
 this canine hdl could be a different lipoprotein from true canine hdl . since the 
 characteristics of this canine lipoprotein remain unclear , so does its role .
furthermore , it 
 is also possible that there would be lipoproteins having both apo b-100 and apo a - i in dogs . 
 quantitative analysis of lipoprotein fractions shows that human ldl particles having apo 
 b-100 have certain amounts of apo a - i and apo e [ 5 , 
 11 ] .
similarly , certain canine particles might have both apo b-100 and apo a - i , 
 although this speculation has yet to be validated .
based on the present results , it is 
 difficult to suggest the u - p method as an accurate method for analysis of canine 
 lipoprotein .
the u - p method can not be a gold standard method for analyzing canine 
 lipoprotein .
lc - ms / ms 
 might be an efficient method for detecting canine apo e. however , analysis by sds - page 
 showed that dogs 13 had different protein amounts in the hdl+ldl , hdl and ldl fractions , 
 indicating that there are individual differences among these dogs .
because the hdl - c and ldl - c concentrations determined by the u - p method correlated well 
 with those determined by gp - hplc , this method may be able to roughly separate canine hdl and 
 ldl , meaning that the u - p method could be useful for detecting alterations in the balance of 
 canine lipoproteins and for clarifying the clinical significance of canine lipoprotein 
 profiles .
however , the ldl / hdl ratio determined by the u - p method was quite different from 
 that determined by gp - hplc . since it is possible that gp - hplc was unable to accurately 
 separate hdl from ldl , it is unclear which of the 2 methods is more accurate at separating 
 canine hdl from ldl . in conclusion
, the present study indicated that the u - p method would be hard to use as a 
 gold standard method for analysis of canine lipoprotein , because it precipitates a certain 
 lipoprotein , which includes apo a - i , with canine ldl .
 abstractdue to the lack of a gold standard method in canine lipoprotein analysis , it is unclear 
 whether canine high - density lipoprotein ( hdl ) and low - density lipoprotein ( ldl ) can be 
 accurately evaluated by the lipoprotein analysis methods used for dogs . 
 this study 
 investigated whether the ultracentrifugation - precipitation ( u - p ) method was suitable as a 
 gold standard method for analyzing canine lipoprotein . 
 first , the u - p method was compared 
 with a gel permeation high - performance liquid chromatography system ( gp - hplc ) . 
 the 
 concentrations of canine hdl cholesterol ( hdl - c ) and ldl cholesterol ( ldl - c ) determined by 
 the u - p method correlated closely with those determined by gp - hplc . however , the canine 
 hdl - c concentration determined by the u - p method was lower than that determined by 
 gp - hplc , and the canine ldl - c concentration determined by the u - p method was higher than 
 that determined by gp - hplc . 
 this study showed that some canine hdl could be precipitated 
 with heparin manganese chloride solution . 
 second , the hdl and ldl fractions separated by 
 the u - p method were analyzed by lc - ms / ms . 
 the hdl fraction was found to contain only 
 apolipoprotein a - i , which is an apolipoprotein of hdl , whereas the ldl fraction contained 
 both apolipoprotein a - i and apolipoprotein b-100 , which is an apolipoprotein of ldl . 
 this 
 data showed that a certain lipoprotein that includes apolipoprotein a - i might precipitate 
 with canine ldl when using heparin manganese chloride solution . 
 these results indicated 
 that the u - p method is not currently a gold standard method for analyzing canine 
 lipoproteins . 
MATERIALS AND METHODS
RESULTS
DISCUSSION