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1 methods Lposcale: a novel advanced lpoproten test based on 2D dffuson-ordered 1 H NMR spectroscopy Roger Mallol, 1, *,, Núra Amgó, *,, Mguel A. Rodríguez,, ** Mercedes Heras,, Mara Vnaxa, *, Núra Plana,, Edmond Rock, Josep Rbalta,, Oscar Yanes, *, Lluís Masana,,, and Xaver Correg *,, Department of Electronc Engneerng,* Unverstat Rovra Vrgl, IISPV, Tarragona, Span ; Spansh Bomedcal Research Centre n Dabetes and Assocated Metabolc Dsorders (CIBERDEM), Madrd, Span ; Bosfer Teslab, Reus, Span ; Centre for Omc Scences (COS)** and Research Unt on Lpds and Atheroscleross, Sant Joan Unversty Hosptal, Unverstat Rovra Vrgl, IISPV, Reus, Span ; and UMMM, INRA-Thex, St. Genes Champanelle, France Abstract Determnaton of lpoproten partcle sze and number usng advanced lpoproten tests (ALTs) s of partcular mportance to mprove cardovascular rsk predcton. Here we present the Lposcale test, a novel ALT based on 2D dffuson-ordered 1 H NMR spectroscopy. Our method uses dffuson coeffcents to provde a drect measure of the mean partcle szes and numbers. Usng 177 plasma samples from healthy ndvduals and the concentraton of ApoB and ApoA from solated lpoproten fractons, our test showed a stronger correlaton between the NMR-derved lpoproten partcle numbers and apolpoproten concentratons than the LpoProfle test commercalzed by Lposcence. We also converted LDL partcle numbers to ApoB equvalents (mllgrams per declter) and our test yelded smlar values of LDL-ApoB to the LpoProfle test (absolute mean bas of 8.5 and 7.4 mg/dl, respectvely). In addton, our HDL partcle number values were more concordant wth the calbrated values determned recently usng on moblty. Fnally, prncpal component analyss dstngushed type 2 dabetc patents wth and wthout atherogenc dyslpdema (AD) on a second cohort of 307 subjects characterzed usng the Lposcale test (area under the curve = 0.88) and showed concordant relatonshps between varables explanng AD. Altogether, our method provdes reproducble and relable characterzaton of lpoproten partcles and t s applcable to pathologcal states such as AD. Mallol, R., N. Amgó, M. A. Rodríguez, M. Heras, M. Vnaxa, N. Plana, E. Rock, J. Rbalta, O. Yanes, L. Masana, and X. Correg. Lposcale: a novel advanced lpoproten test based on 2D dffuson-ordered 1 H NMR spectroscopy. J. Lpd Res : Supplementary key words low densty lpoproten partcle number cardovascular rsk nuclear magnetc resonance two-dmensonal Abnormal levels of blood lpds, such as hgh concentratons of LDL cholesterol (LDL-C) and low concentratons of HDL cholesterol (HDL-C), ncrease the rsk of CVD, the frst cause of death n developed countres ( 1 ). Accordngly, the European Atheroscleross and Cardology Socetes and the Natonal Cholesterol Educaton Program, through ther thrd report of the Adult Treatment Panel, establshed LDL-C and nonhdl-c as the prmary and secondary target, respectvely, of cholesterol-lowerng therapy to reduce cardovascular rsk ( 2 ). LDL-C-lowerng therapy has been shown to reduce the rate of cardovascular events n patents wth or wthout cardometabolc rsk (CMR) ( 3, 4 ). However, a large proporton of patents under treatment, as well as undagnosed ndvduals, may suffer cardovascular events despte showng normal LDL-C levels. Cardovascular events are more lkely to occur n patents wth dabetes and metabolc syndrome. These pathologes share a common phenotype characterzed by a hgh content of trglycerdes, a preponderance of small dense LDL partcles, and low HDL levels. In ndvduals wth ths partcular phenotype, LDL-C has been shown to be a poor predctor of cardovascular rsk, so standard Ths work was partally funded by CIBER de Dabetes y Enfermedades Metabólcas, an ntatve of ISCIII (Mnstero de Cenca e Innovacón), as well as the FIS (Project PI081579). Ths work was also partly supported by the EU FP5 Program Qualty of Lfe and Management of Lvng Resources, Key Acton 1, Food, Nutrton, and Health, enttled Vtamn A, Vtamn E, and Carotenod Status and Metabolsm durng Ageng: Functonal and Nutrtonal Consequences, acronym VITAGE (Contract QLK1-CT ). R.M. and N.A. are employed by or have leadershp roles n Bosfer Teslab. X.C. and L.M. have advsory roles at Bosfer Teslab. R.M., N.A., X.C., and L.M. are stock owners of Bosfer Teslab. Manuscrpt receved 14 Aprl 2014 and n revsed form 5 January Publshed, JLR Papers n Press, January 7, 2015 DOI /jlr.D Abbrevatons: AD, atherogenc dyslpdema; ALT, advanced lpoproten test; AUC, area under the curve; CMR, cardometabolc rsk; CV, coeffcent of varaton; DOSY, dffuson-ordered 1 H NMR spectroscopy; FDA, US Food and Drug Admnstraton; HDL-C, HDL cholesterol; HDL-P, HDL partcle number; LDL-C, LDL cholesterol; LDL-P, LDL partcle number; PCA, prncpal component analyss; PE, percentage error; PLS, partal least squares; ROC, recever-operatng curve; T2DM, type 2 dabetc; VLDL-P, VLDL partcle number. 1 To whom correspondence should be addressed. e-mal: rmallol@bosferteslab.com The onlne verson of ths artcle (avalable at contans supplementary data n the form of text, two fgures, and three tables. Copyrght 2015 by the Amercan Socety for Bochemstry and Molecular Bology, Inc. Ths artcle s avalable onlne at Journal of Lpd Research Volume 56,

2 lpd panels that measure the cholesterol or trglycerde content of lpoprotens seem to be nsuffcent to predct rsk of CVD. To fll ths gap, advanced lpoproten tests (ALTs) ( 5 ) that allow for an extensve characterzaton of lpoproten partcles through a range of addtonal parameters, such as sze and partcle number, have been proposed for mprovng assessment of rsk of CVD and for gudng lpd-lowerng therapes ( 6 ). NMR spectroscopy s a technque that enables analyss of lpoproten partcles ( 5 ). Brefly, dependng on the sze of the partcle, the methyl moetes of the lpds n lpoproten partcles resonate at slghtly dfferent frequences, the smaller partcles resonatng at lower frequences. Therefore lpoprotens can be quantfed ether by decomposng the methyl sgnal of the core lpds nto ndvdual sgnals ( 7, 8 ) or usng statstcal methods on the entre methyl envelope to estmate lpd concentratons ( 9 ). Currently three methodologes use NMR to characterze lpoproten partcles. The method descrbed by Jeyarajah, Cromwell, and Otvos ( 7 ) provdes the sze and partcle number of the man lpoproten classes (.e., VLDL, LDL, and HDL) and the partcle number of nne lpoproten subclasses. Ths method s bult on a lbrary of 1D NMR spectra from prevously solated lpoproten fractons and on an algorthm that fts ther NMR methyl sgnal wth those of lpoprotens from serum or plasma samples. The partcle szes of the solated lpoproten fractons were determned by transmsson electron mcroscopy and gradent gel electrophoress. A second method descrbed by Kaess et al. ( 8 ) characterzes ffteen lpoproten subclasses measurng the samples by magnetc feld gradent ntenstes and temperatures. The latter NMR methodology was reported by Ala- Korpela and colleagues ( 9 ), and t estmates lpd content, sze, and partcle numbers of the man lpoproten classes, as well as the partcle numbers of fourteen lpoproten subclasses based on regresson models calbrated usng the lpd content and sze obtaned by hgh performance lqud chromatography. NMR-based ALTs have demonstrated that LDL and HDL partcle numbers (LDL-P and HDL-P) are more powerful than classcal cholesterol markers as ndces of cardovascular rsk ( 10 ). For nstance, LDL-P better ndcated atherosclerotc rsk than LDL-C n ndvduals wth dscordant LDL-P and LDL-C levels ( 11 ). Ths dscordance s usually explaned based on the large varablty n the amount of cholesterol per LDL partcle and to dfferences n LDL partcle sze. In another study, HDL-P, but not HDL-C, was nversely assocated wth carotd ntma-meda thckness after adjustng for covarates ( 12 ). Moreover, the use of NMR-derved lpoproten subclasses mproved rsk stratfcaton for subclncal atheroscleross n comparson to conventonal lpds ( 13 ). Altogether, these and other evdence have precptated two sgnfcant events: 1 ) the 510(k) clearance from the US Food and Drug Admnstraton (FDA) to market the Vantera Clncal Analyzer commercalzed by Lposcence, the frst NMR-based dagnostc platform that determnes LDL-P; and 2 ) the recommendaton from the Amercan Assocaton for Clncal Chemstry Lpoproten and Vascular Dseases Dvson Workng Group on Best Practces to nclude LDL-P n the gudelnes used to manage CMR based on the evdence collected from 25 clncal studes ( 14 ). Despte these advances, there s stll some controversy about the ntroducton of NMR-based ALTs nto clncal practce, partly due to the fact that current methods do not provde a drect measure of lpoproten szes. As an alternatve to current NMR methods, here we present the Lposcale test, a novel method for characterzng lpoproten partcles based on 2D dffuson-ordered 1 H NMR spectroscopy (DOSY). DOSY allows measurng the dffuson coeffcents and drectly calculatng lpoproten szes through the Stokes-Ensten equaton ( 15 ). It s noteworthy that a drect measure of lpoproten szes s of partcular mportance because they are used to compute lpoproten partcle numbers by dvdng the spatal volume of the total lpd molecules by the mean volume (.e., sze) of the lpoproten partcles. Our ratonale s that usng DOSY to drectly calculate lpoproten szes should yeld more accurate measurements of lpoproten partcle numbers than current NMR-based ALT methods. To develop our new DOSY-based ALT, we used a cohort of 177 healthy ndvduals and then we compared the lpoproten partcle numbers obtaned usng the Lposcale test wth those obtaned usng the FDA-cleared ALT commercalzed by Lposcence. Fnally, we appled the Lposcale test to characterze a second cohort of 307 type 2 dabetc (T2DM) patents wth and wthout atherogenc dyslpdema (AD). Our results demonstrate that our methodology can be appled to study samples wth aberrant lpd and lpoproten concentratons and can add nsght nto the understandng of metabolc dseases. MATERIALS AND METHODS Study subjects We used samples from the VITAGE project to develop the Lposcale test ( 16 ). Brefly, 177 healthy nonsmokng men (0 cgarettes/day for >6 months) were enrolled n Clermont-Ferrand (France) and Reus (Span). Excluson crtera were famlal hypercholesterolema, chronc dseases (ncludng dabetes, cancer, cardac nsuffcency, nflammatory dseases, and unstable hypertenson), or alcohol abuse. Mean age was 45.8 ± 15.5 years, ncludng a mnmum age of 19 years and a maxmum age of 75 years. Fastng venous blood samples were collected n EDTA tubes and centrfuged mmedately for 15 mn at 4 C at 1,500 g. Plasma samples were then kept at 80 C untl further analyss. The ethcs commttee of the two recrutng centers approved the study protocol, and wrtten nformed consent was obtaned from all volunteers. We used a second cohort to valdate the results provded by the Lposcale test. Elgble partcpants were 307 T2DM men and women, rangng from 30 to 80 years of age, wth (n = 91) or wthout (n = 216) AD. AD was defned as havng trglycerde levels over 150 mg/dl and HDL-C levels under 40 mg/dl (men) or 50 mg/dl (women). Excluson crtera were the presence of CVD and chronc hepatc or renal alteratons. Patents under lpdlowerng medcaton entered a wash-out perod of 4 weeks. Samples were kept at 80 C untl NMR analyss. The study protocol was also approved by the ethcs commttee of the partcpatng nsttuton. 738 Journal of Lpd Research Volume 56, 2015

3 Lpd and lpoproten measurements Lpoprotens were separated by sequental preparatve ultracentrfugaton, usng a Kontron 45.6 fxed-angle rotor n a Centrkon 75 (Kontron Instruments, Italy). The lpoproten fractons solated were VLDL (d < g/ml), IDL (d = g/ml), LDL (d = g/ml), and HDL (d = g/ml). Ther cholesterol and trglycerde content was quantfed usng standard enzymatc assays adapted to the Cobas-Mra-Plus autoanalyzer (SPINREACT S.A.U., Span) ( 17 ). The concentraton of ApoB100 and ApoA1 n the ultracentrfuged fractons was also quantfed usng an mmunoturbdmetrc assay adapted to the Cobas-Mra-Plus auto-analyzer (SPINREACT S.A.U.). The assay and the value of the calbrator concentraton were standardzed aganst the Certfed Reference Materal ApoA1 WHO/IFCC SP1-01 and ApoB100 WHO/ IFCC SP3-07. Plasma samples were also analyzed by Lposcence (Ralegh, NC) to obtan reference values of VLDL, LDL, and HDL szes and partcle numbers ( 7 ). Advanced lpoproten testng usng DOSY 2D DOSY. Plasma samples were analyzed by NMR spectroscopy usng a modfed exstng protocol ( 18 ). Brefly, 1 H NMR spectra were recorded on a BrukerAvance III 600 spectrometer, operatng at a proton frequency of MHz (14.1 T), at 310 K. We used the double stmulated echo pulse program wth bpolar gradent pulses and a longtudnal eddy current delay. The relaxaton delay was 2 s, the fnte mpulse decays were collected nto 64,000 complex data ponts and 32 scans were acqured on each sample. The gradent pulse strength was ncreased from 5 to 95% of the maxmum strength of 53.5 Gauss cm 1 n 32 steps. The squared gradent pulse strength was lnearly dstrbuted. Surface fttng. The methyl sgnal was surface ftted usng a prevously reported procedure ( 18 ). The number of functons was ncreased to account for the nne lpoproten subclasses. The unqueness of the solutons was studed by fttng each sample 10 tmes wth randomly chosen ntal values of the sgnal ntenstes. As a result, we obtaned unque solutons for all samples after 10 runs. Normalzed root mean square errors of the fttngs were calculated as prevously descrbed ( 18 ). The ntal values of the sgnal ntenstes were taken to be the mean soluton values of all samples. Predcton of lpd concentraton. Partal least squares (PLS) regresson models were calbrated to predct the cholesterol and trglycerde concentraton of the man lpoproten fractons (VLDL, LDL, and HDL). Valdaton performance of the PLS models were assessed by venetan blnds cross-valdaton splttng the data 10 tmes. Coeffcents of determnaton between the predcted and reference concentratons ranged from 0.79 to 0.98 n the calbraton step. The coeffcents of determnaton of the valdaton step ranged from 0.81 to 0.98 and are n the range of other reported studes ( 19 ). Lpoproten szng and partcle number determnaton. The NMR functons were assocated wth a gven lpoproten class (VLDL, LDL, or HDL) accordng to ther assocated NMR sze. The man lpoproten fractons were defned as VLDL ( nm), LDL ( nm), and HDL ( nm). The mean partcle sze of every man fracton (VLDL, LDL, and HDL) was derved by averagng the NMR area of each fracton by ts assocated sze. To obtan partcle-weghted lpoproten szes, each NMR area was dvded by ts assocated volume. Then a mean partcle sze was obtaned for each lpoproten class by multplyng the NMR lpoproten partcle szes by ther fractonal partcle concentraton relatve to the total partcle concentraton of a gven class : n A d = 1 3 d VLDL sze nm =, = 1,,3 n A = 1 3 d n A d = 1 d 3 LDL sze nm =, = 4,, 6 n A = 1 3 d n A d = 1 d 3 HDL sze n m =, = 7,, 9 n A = 1 3 d ( Eq. 1 ) where A and d are the area (au) and dameter (nm) of a gven lpoproten partcle. The partcle numbers of each lpoproten man fracton were calculated by dvdng the lpd volume by the partcle volume of a gven class. The lpd volumes were determned by usng common converson factors to convert concentraton unts obtaned from the PLS models nto volume unts ( 7 ). The relatve areas of the lpoproten components used to decompose the 2D spectra were used to derve the partcle numbers of the nne lpoproten subclasses. Spkng experments We performed spkng experments to valdate the groupng of the NMR functons. Serum samples were obtaned from three volunteers. Lpoproten fractons (VLDL, LDL, and HDL) were obtaned by sequental ultra-centrfugaton as descrbed above. Then, serum samples were spked wth the lpoproten fractons from the same subjects one by one. A serum sample made up wth buffer to equal volumes as the spked samples was also analyzed. Analytcal performance The analytcal performance of the Lposcale test nvolved blood samples from two volunteers. Brefly, fastng venous blood samples were collected n EDTA tubes and centrfuged mmedately for 15 mn at 4 C at 1,500 g. Then, fve alquots were obtaned for each subject and kept at 80 C untl the NMR analyss. The wthn-assay precson of the method was studed based on the analyses of the fve alquots of the two subjects n the same day, whle the nterassay precson was based on the analyses of the fve alquots of the two subjects through three consecutve days. Statstcal analyses The Bland-Altman plot was used to measure the agreement between both NMR technques n assessng partcle szes and numbers ( 20 ). Ths plot uses the dfferences between observatons made by the two methods on the same subjects and t analyzes the agreement between two methods n terms of bas and precson. When there s a relatonshp between dfference and magntude, the standard Bland-Altman analyss can be extended ether wth a logarthmc transformaton approach or a more general regresson approach. As a measure of agreement, we calculated the percentage error (PE) between both technques, as descrbed prevously ( 21 ): Lposcale: an advanced lpoproten test based on DOSY 739

4 2 2 PE CVnew CV old (%) = + ( Eq. 2 ) where the coeffcent of varaton (CV) [CV (%) = ( / ) 100] s defned as the rato of the standard devaton to the mean of the populaton. We also defned a maxmum PE by usng the CV of the old method twce n equaton 2. Spearman analyss was used to assess the relatonshp between contnuous varables. All the analyses were performed wth MAT- LAB verson R2010a (MathWorks). PLS and prncpal component analyss (PCA) models were bult usng PLS Toolbox (Egenvector Research). RESULTS Assgnment of DOSY NMR functons to the man lpoproten classes The Lposcale test provdes lpd concentratons (.e., trglycerdes and cholesterol), szes, and partcle numbers for VLDL, LDL, and HDL classes, as well as the partcle numbers of nne subclasses, namely large, medum, and small VLDL, LDL, and HDL, respectvely. Fgure 1 provdes an overvew of the Lposcale test showng the most mportant processes on whch the characterzaton of lpoproten classes s based. The use of the dffuson dmenson Fg. 1. Overvew of the Lposcale test. A: Expermental DOSY spectra. Seven gradents out of 32 total are shown for vsualzaton purposes. The frst gradent has been flled n black to show the NMR regons of the spectra that are used to calbrate and predct cholesterol and trglycerde concentratons usng PLS regresson. In red, we show the attenuaton of the methyl NMR sgnal along the gradent axs to show the regon used to characterze the dfferent lpoproten subclasses. B: Surface fttng process for a gven subject. The expermental attenuaton of the methyl NMR sgnal (left) and the nne NMR functons used to ft the expermental surface (rght). C: Usng ths methodology, we can elucdate nne lpoproten subclasses, namely large, medum, and small VLDL, LDL, and HDL. 740 Journal of Lpd Research Volume 56, 2015

5 represents the man dfference between our approach and the NMR-based platform commercalzed by Lposcence. The Lposcale test uses 2D spectra from DOSY experments ( Fg. 1A ) to decompose the (CH 3 ) proton resonances of the lpds n lpoproten partcles nto nne Lorentzan functons (.e., F1 to F9) ( Fg. 1B ). Of note, the cholesterol esters and trglycerdes n the partcle core contrbute wth three methyl groups each ( 5 ) and thus the total amount of all the methyl groups s mostly dependent on partcle sze (see Dscusson). Ths approach largely prevents multple solutons and, consequently, enhances robustness of the measurements. Indeed, we obtaned an average normalzed root mean square error for the surface fttngs of less than 1.85% (see the Materals and Methods for detals), whch ndcates that our deconvoluton s hghly reproducble. Accordng to the Ensten-Stokes equaton, the larger the dffuson coeffcent, the smaller the sze of a gven lpoproten partcle wll be. Thus, we assocated the nne NMR functons wth the three man lpoproten classes based on ther descrbed sze ranges ( Fg. 1C ) ( 22 ): F1 to F3 ( nm) were assocated wth the VLDL partcles, functons F4 to F6 ( nm) wth the LDL partcles, and functons F7 to F9 ( nm) wth the HDL partcles. These assocatons were valdated usng spke-n experments. We solated VLDL, LDL, and HDL partcles from serum samples of three volunteers by ultracentrfugaton (see the Materals and Methods for detals), and each solated lpoproten fracton was mxed one by one wth the serum sample of the same ndvdual. Fgure 2 shows the average relatve change of every lpoproten class n serum, represented as the total NMR area correspondng to VLDL (.e., F1 to F3), LDL (F4 to F6), and HDL (F7 to F9) partcles by applyng our surface-fttng algorthm. Our results ndcate that only the area of the lpoproten fracton that has been spked-n shows a substantal ncrement, demonstratng that our NMR functons are correctly assgned to VLDL, LDL, and HDL partcles. Correlaton between lpoproten partcle numbers and apolpoproten concentratons Next, we explored the degree of correlaton between the VLDL-, LDL-, and HDL-Ps calculated usng the Lposcale and the LpoProfle tests, and the apolproproten content of each lpoproten class, because the apolpoproten concentratons n each class can serve as a surrogate of the number of partcles. Fgure 3A shows a strong Fg. 2. Spke-n experments wth solated VLDL, LDL, and HDL partcles n serum samples. Mean relatve changes n the total area under the NMR functons for each lpoproten class (VLDL = F1 + F2 + F3, LDL = F4 + F5 + F6, and HDL = F7 + F8 + F9). Each color represents spked serum samples ether wth solated VLDL, LDL, or HDL fractons. Fg. 3. Regresson analyses that examne the relatonshp among lpoproten partcle numbers usng the Lposcale and LpoProfle tests, and the apolpoproten concentratons of VLDL (A), LDL (B), and HDL (C) partcles. Lposcale: an advanced lpoproten test based on DOSY 741

6 postve lnear relatonshp between the concentraton of VLDL-ApoB determned usng bochemcal methods and the VLDL-P values determned usng the Lposcale test ( r = 0.91) and the LpoProfle test ( r = 0.75). Smlarly, the correlaton coeffcents between LDL-P determned va the Lposcale and LpoProfle tests and ther respectve apolpoproten concentraton were 0.82 and 0.78, respectvely ( Fg. 3B ). Fnally, the correlaton coeffcents between HDL-P and HDL-ApoA were 0.65 for the Lposcale test and 0.60 for the LpoProfle test ( Fg. 3C ). Thus, the Lposcale test showed a stronger correlaton between lpoproten partcle number and apolpoproten concentraton than the FDA-cleared NMR method that we took as the benchmark for the measurement of the number of partcles n each lpoproten class. Fnally, we evaluated the effects of dfferent proportons of trglycerdes and cholesterol concentratons wthn the LDL lpoproten partcles on the correlatons between LDL-ApoB measures and LDL-P concentratons. Subjects were dvded nto tertles of the LDL-trglycerde to LDL-C rato and the correlatons between LDL-ApoB and LDL-P values determned by the two technques were evaluated (see supplementary Fg. 1). The correlaton coeffcents between LDL-P determned va the Lposcale test and ts respectve apolpoproten concentraton ncreased across tertles (0.84, 0.86, and 088, respectvely), whle the Lpo- Profle test showed decreasng correlaton coeffcents across tertles (0.83, 0.79, and 0.76, respectvely). LDL partcle sze and number agreement between the Lposcale and the LpoProfle NMR tests LDL-P s the most valdated and clncally useful parameter that ALTs can determne. In ths regard, three consensus reports ( 3, 23, 24 ) have all recommended that ApoB and/or measurements of LDL-P shall be ncorporated nto exstng consensus gudelnes for advanced CMR management. Thus, here we measured the agreement between our 2D-NMR test (.e., Lposcale) and the 1D-NMR test (.e., LpoProfle ) (see Fg. 4A, B ). From the VITAGE cohort, the mean LDL-sze and LDL-P were Fg. 4. Bland-Altman analyses to measure the agreement between both NMR tests n assessng partcle szes and numbers. Comparson of LDL-sze (A) and LDL-P (B) as assessed by the Lposcale and the LpoProfle tests. Comparson of LDL-ApoB concentratons measured from solated LDL fractons usng a classcal bochemcal method and the same concentraton estmated usng the Lposcale test (C) and the LpoProfle test (D). 742 Journal of Lpd Research Volume 56, 2015

7 20.6 nm and 843 nmol/l usng Lposcale, and 21.3 nm and 1,133.1 nmol/l usng LpoProfle, respectvely. Ths results n an average LDL-sze and LDL-P dfference (.e., bas) between both methods of 0.7 nm and nmol/l, respectvely. We further analyzed the agreement between both methods usng the Bland-Altman plot. The plot s characterzed by a proportonal bas n the form of a negatve lnear relatonshp between the two methods resultng from fewer small LDL partcles usng the Lposcale test n comparson wth the LpoProfle test. The logarthmc transformaton of data dd not correct ths proportonal error. Thus, to narrow down the lmts of agreement, the dfference between the methods was regressed on the average of the two methods ( Fg. 4B ). The CVs of LDL sze and LDL-P were 3.14 and 31.0%, respectvely, usng the Lposcale test, and 3.4 and 32.3%, respectvely, usng the LpoProfle test. The PE obtaned between both methods was 4.6% for LDL sze and 44.7% for LDL-P. In short, we consder that the agreement between both NMR tests s acceptable based on a PE below 5% for LDL-sze and 50% for LDL-P. The concentraton of ApoB100 serves as a reference value of LDL-P due to the fact that each LDL partcle contans only one molecule of ApoB100. In ths regard, some authors have suggested that LDL-Ps (nanomoles per lter) should be converted to ApoB equvalents (mllgrams per declter) to allow a drect comparson wth an establshed bochemcal parameter ( 14 ). Thus, to nvestgate whch NMR test yelded more accurate LDL-P values, we converted LDL partcle concentraton (nanomoles per lter) nto ApoB concentraton (mllgrams per declter) on the bass of a molecular mass for ApoB100 of 550 kda ( 14 ). Once agan, we assessed the agreement between the LDL- ApoB concentratons estmated by the Lposcale and LpoProfle tests, and the same concentraton assayed bochemcally from LDL fractons solated by ultracentrfugaton (see Materals and Methods for detals) usng the Bland-Altman plot. The Bland-Altman analyss revealed a mean dfference between the LDL-ApoB concentraton estmated usng the LpoProfle test and bochemcal methods of 7.4 mg/dl, whle for Lposcale ths mean dfference was 8.5 mg/dl ( Fg. 4C, D ). The CV of LDL- ApoB concentraton bochemcally assayed was 26.5%, yeldng a maxmum PE of 40%. LDL-ApoB concentraton obtaned usng the Lposcale and the LpoProfle tests yelded CVs of 31.0 and 32.3%, respectvely. Thus, the PE between the bochemcal assay and Lposcale was 40.8%, and 41.8% when comparng wth LpoProfle. 5%. The nter-assay precson on the same parameters, calculated from the same fve alquots run on three consecutve days, was 8% (see supplementary Tables 1, 2). Smlarly, the wthn-assay and nter-assay precson for cholesterol and trglycerde concentraton, and VLDLand HDL-P, were 6%. Fnally, both wthn-assay and nter-assay precson for the mean partcle sze for every lpoproten class were 1%. Overall, the precson values of our 2D-NMR method are wthn the range of those reported by Lposcence Inc. for the LpoProfle test ( 7 ). Characterzaton of AD n an ndependent cohort of dabetc patents To demonstrate that the Lposcale test can be used to characterze and dscrmnate ndvduals wth aberrant lpd and lpoproten values, we mplemented our 2D- NMR method on a cohort study of 307 subjects that ncluded T2DM patents wth (n = 91) and wthout (n = 216) AD. A PCA usng the nput varables cholesterol and trglycerde concentratons, mean szes, and mean partcle numbers of VLDL, LDL, and HDL classes, and partcle numbers of nne subclasses of lpoprotens, shows two separate clusters along PC1 (43.81% of the varance) correspondng to the two patent groups, that s, T2DM and T2DM wth AD ( Fg. 5A ). In order to evaluate whch are Analytcal performance of the Lposcale test Next we assessed the precson of the Lposcale test understood as the ablty of the assay to consstently reproduce the same result when dfferent sample alquots are taken from the same specmen. The wthn-assay precson of the method, calculated from the analyss of fve dfferent alquots from two dfferent subjects wthn the same day (.e., n = 10), on the determnaton of cholesterol and trglycerde concentratons, and partcle numbers for the LDL class and ts small, medum, and large subclasses, was Fg. 5. Scores plot (A) and loadngs plot (B) for a PCA model bult to dscrmnate between T2DM wth and wthout AD. Lposcale: an advanced lpoproten test based on DOSY 743

8 the man varables responsble for the separaton of the T2DM patents wth and wthout AD, the resultng loadngs were vsualzed n a varables-loadng plot ( Fg. 5B ). The plot reveals that the trad of AD, namely ncreased blood concentratons of small dense LDL partcles, decreased HDL partcles, and ncreased trglycerde concentraton, contrbutes to the separaton of the two patent groups. Importantly, the loadngs plot also shows that T2DM patents wth AD have a smaller number of large and medum LDL and HDL partcles relatve to T2DM patents wthout AD. Moreover, patents wth T2DM and AD have a greater number of total LDL and, n partcular, of small LDL partcles. Consderng the frst prncpal component as the output of a putatve classfer, we calculated the recever-operatng curve (ROC) n order to evaluate the classfcatory power of our PCA model. As can be observed n the ROC curve depcted n Fg. 6, our approach showed an area under the curve (AUC) of 0.88, showng an excellent classfcaton performance to dscrmnate between T2DM patents wth and wthout AD. All together, these results demonstrate that the Lposcale test s clncally useful to classfy ndvduals showng an abnormal lpd and lpoproten pattern that s typcal of AD, whch has emerged as an mportant rsk factor for myocardal nfarcton and CVD. Fnally, we valdated the correlatons between ApoB measures and lpoproten partcle concentratons at dfferent trglycerde to cholesterol ratos on a group of 22 T2DM subjects wth AD treated for 12 weeks wth both fenofbrate and/or nacn (see supplementary Table 1) because these treatments may change the proporton of trglycerde and cholesterol concentratons wthn the lpoproten partcles. In ths study, we used the total plasma concentraton of ApoB as a reference value of total ApoB-contanng lpoproten partcle concentraton. Thus, we summed the partcle numbers of the VLDL and LDL classes to obtan the total ApoB-contanng lpoproten partcle numbers and then we converted these values nto ApoB equvalents as descrbed Fg. 6. ROC curve evaluatng the dscrmnatory power of the PCA model. before. The results of the correlaton analyss show a smlar hgh degree of correlaton between the sum of the concentratons of ApoB of the dfferent lpoproten fractons obtaned by NMR and the total concentraton of ApoB obtaned bochemcally at baselne (supplementary Fg. 3A, r = 0.83), after the fenofbrate nterventon (supplementary Fg. 3B, r = 0.88), and after the nacn nterventon (supplementary Fg. 3C, r = 0.89). DISCUSSION The methyl moetes of the lpds n lpoproten partcles resonate at slghtly dfferent frequences dependng on the sze of the partcle, the smaller partcles resonatng at lower frequences ( 25 ). Though dfferent lpoproten subclasses may n theory be deconvoluted wthn the 1D- NMR spectrum accordng to ther chemcal shft postons, t has proven to be complcated n practce due to sgnfcant spectral overlap as dfferent lpoproten subclasses contrbute to NMR resonance at the same frequency ( 18 ). Ths s of great mportance for LDL subclasses, whch are partcularly dffcult to characterze due to the effect of neghborng lpoproten classes (.e., VLDL and HDL) ( 26, 27 ). Moreover, because the cholesterol esters and trglycerdes n the partcle core contrbute wth three methyl groups each, the total amount of all the methyl groups s not affected by varatons n core lpd compostons,.e., varatons n the cholesterol ester to trglycerde rato drven by the cholesterol ester transfer proten (CETP), but s mostly dependent on partcle sze ( 28 ). Thus, the addton of a second dmenson n the NMR spectrum by means of a dffuson experment (.e., DOSY) helps to better characterze the dfferent lpoproten subclasses. DOSY allows the separaton of the lpoproten subclasses accordng to ther dffuson coeffcent, and wth the use of the Stokes-Ensten equaton, DOSY NMR yelds an objectve separaton of lpoproten subclasses based on ther sze and favors the unqueness of mathematcal solutons compared wth 1D-NMR. In ths study we present a novel ALT based on DOSY NMR spectroscopy called Lposcale, whch has been compared wth the establshed NMR method developed and commercalzed by Otvos and colleagues and Lposcence (.e., LpoProfle ) ( 7 ), respectvely, whch has become the only FDA-cleared blood test that drectly quantfes LDL partcles. Our study consttutes the frst attempt to compare two 1 H NMR methodologes, whch s partcularly mportant wthn the framework of standardzed ALTs ( 14, 29 ) f LDL-P s ntended to be used n cardovascular rsk management. The agreement between the 2D-NMR (Lposcale) and 1D-NMR (LpoProfle ) methods for measurng LDL-P revealed a lnear dependency between the dfference and the magntude of the measurements. However, the LDL- ApoB values obtaned usng our 2D-NMR method were more n conformty wth bochemcal values than those obtaned usng the FDA-cleared test. Ths dsagreement between the two NMR methodologes may come partly 744 Journal of Lpd Research Volume 56, 2015

9 from the small bas between the lpoproten szes determned by the two methods, through whch our 2D-NMR method estmates smaller LDL partcle szes n compar son wth the 1D-NMR method. Ths mght be a consequence of the method used to derve the partcle concentratons. Whle the LpoProfle test computes massweghted partcle numbers, our method computes partcleweghted partcle numbers. From our pont of vew, the partcle-weghted approach s more realstc because the concentraton of small partcles exceeds the concentraton of larger ones. A prevous study also reported a lnear dependence between the dfference and magntude of LDL-sze measured by Lposcence and gradent gel electrophoress ( 30 ). On the other hand, no method-comparson study has evaluated dfferent LDL-P assays. As well as the Lposcence test, on moblty analyss has been shown to be an alternatve method for drectly measurng LDL-P ( 31 ). Although concerns were rased about ts lack of accuracy ( 32 ), the authors further ncluded correcton factors whch corrected the measurement of concentraton of both non- HDL and HDL partcles ( 33 ). Concernng HDL partcles, our 2D-NMR method showed a better correlaton between HDL-P and HDL- ApoAI concentraton relatve to the LpoProfle test. Even so, regardless of whch NMR method s used, the correlaton between HDL-P and HDL-ApoAI s not as good as when correlatng LDL-P and the ApoB content. Ths could be partally explaned by the lack of a drect relatonshp between HDL partcles and ApoAI molecules. Unlke n LDL where each partcle contans one molecule of ApoB, HDL partcles contan between 2 and 4 molecules of ApoAI. A recent study has shown that the values of HDL-P estmated usng an updated verson of the Shen model, that relates the HDL-sze and the rato of HDL-C to ApoAI concentraton, are approxmately 50 60% lower than the HDL-P measurements obtaned usng the establshed NMR technque commercalzed by Lposcence ( 34 ). Importantly, as can be seen n Fg. 3C, the Lposcale test estmated HDL-P values that are lower than the ones estmated by the LpoProfle test, and consequently are more concordant wth the number of HDL partcles estmated by the modfed Shen model and the calbrated on moblty methodology reported recently by Hutchns et al. ( 34, 35 ). Fnally, the characterzaton of IDL lpoprotens by NMR spectroscopy s not straghtforward due to ) ts low concentraton range compared wth the other lpoprotens, and ) ts NMR response arses between the small VLDL and large LDL lpoprotens n terms of chemcal shft. Takng ths nto account, we chose to be cautous and avod the characterzaton of IDL, even n the event that ths could appear as a lmtaton of our method. However, the solaton of the IDL fractons to obtan pure VLDL and LDL fractons allowed for a good characterzaton of the latter, whch n turn are the most clncally useful together wth the HDL fracton. Moreover, although n subjects wth normal lpd levels the concentratons of VLDL- ApoB and IDL-ApoB mght be low and therefore below the lmt of detecton of the mmunoturbdmetrc assay, n ths crcumstance the clncal utlty of these parameters s mnmal. However, n T2DM subjects or patents wth AD, the concentratons of VLDL-ApoB and IDL-ApoB are hgher and clncally relevant, and n ths case the technque s very robust. CONCLUSIONS We evaluated a new methodology for quantfyng lpoproten subclasses based on 2D DOSY, whch drectly measures the szes of lpoproten partcles. 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