Biomarkers for response to bariatric surgery

Abstract

The invention relates to the in vitro use of various molecular biomarker levels measured in samples from subjects with obesity, said samples being isolated before the bariatric surgery as biomarkers for predicting or prognosing the response to bariatric surgery.

Description

[0001] BIOMARKERS OF RESPONSE TO BARIATRIC SURGERY

[0002] DESCRIPTION

[0003] TECHNICAL FIELD

[0004] The present invention falls within the field of medicine, specifically the field of medical prognosis and diagnosis, and even more specifically refers to the use of biomarkers to predict the response of subjects undergoing bariatric surgery.

[0005] STATE OF THE PRIOR ART

[0006] Obesity is a chronic disease characterized by an increase in body adipose tissue and a chronic, low-grade inflammation that predisposes the individual to the development of a range of associated comorbidities, such as dyslipidemia, high blood pressure, and / or type 2 diabetes, which are all included in the so-called metabolic syndrome.

[0007] Bariatric surgery is currently the most effective treatment for the long-term control of morbid obesity and metabolic syndrome, surpassing dietary therapy, lifestyle modifications, exercise, behavioral therapy, and drug treatment, as these types of therapies have high recurrence rates and insufficient weight loss to significantly improve comorbidities. Although it is an invasive procedure, bariatric surgery has proven to be a safe and effective technique, currently showing a mortality rate of less than 0.5%. The main benefit of bariatric surgery is a significant decrease in body weight, accompanied by a decrease in adipose tissue and an improvement in associated comorbidities, primarily related to carbohydrate metabolism.However, it can be associated with a number of potentially serious complications, both early (anastomotic leaks, gastrointestinal bleeding, deep vein thrombosis, pulmonary thromboembolism, intestinal obstruction, surgical incision infection, pneumonia and cardiac events) and late (marginal ulcers, internal hernias, dumping syndrome, cholecystitis, anemia and osteomalacia).

[0008] Wide variability has been observed in the response of subjects to surgery, with the same benefits in improving obesity and its comorbidities not being achieved in all patients. Some subjects experience insufficient weight loss and reduction of cardiovascular risk factors, as well as persistent type 2 diabetes. The exact causes of this resistance to bariatric surgery are unknown, although many preoperative factors (negative or positive) have been proposed as predictors based on body weight reduction or the remission of diabetes after surgery. Some factors that may promote its onset have also been identified, such as age and plasma levels of inflammatory cytokines.However, none of these factors have consistently been shown to have sufficiently strong predictive power to influence eligibility for bariatric surgery per se, and no molecular markers predictive of bariatric surgery have been identified that would allow for the design of more specific inclusion criteria and strategies.

[0009] Therefore, it would be very useful to have predictive response markers for weight loss induced by bariatric surgery to ensure a suitable situation for each subject. To date, the initial state of adipose tissue, which could be a key factor in the variability of the observed response, has not been taken into account. Furthermore, since adipose tissue is the main organ affected in bariatric surgery, as the majority of the percentage of loss associated with the intervention is due to a decrease in this tissue, knowledge of the prior state of adipose tissue before the intervention could be considered a criterion for evaluating and selecting subjects as candidates for bariatric surgery.Therefore, having factors that predict response to bariatric surgery based on the initial state of adipose tissue could be a key tool in clinical practice, allowing for improved subject selection by identifying those who should receive more extensive follow-up or alternative therapies.

[0010] The most comprehensive approach to this goal comes through the field of proteomics. Because proteins reflect underlying cellular biological processes, studying the adipose tissue proteome could allow for the exploration of the biological and pathophysiological mechanisms involved in the relationship between obesity and its associated comorbidities. The use of "molecular fingerprints" obtained from proteomic studies are widely used and very useful tools in the diagnosis of numerous pathologies. Thus, the identification of a molecular fingerprint capable of predicting adipose tissue status could be a potential preoperative prediction tool that, together with other established criteria for bariatric surgery, can reduce variability in the improvement response, thereby increasing the success rates of this intervention.

[0011] DESCRIPTION OF THE INVENTION The object of the present invention is to establish preoperative parameters that allow predicting the capacity for response to bariatric surgery for weight loss. In this regard, a preferred molecular biomarker, CYB5 (cytochrome b5, form A), and a panel of molecular biomarkers that includes it have been defined. The preoperative expression levels of the biomarkers in subcutaneous adipose tissue allow predicting the response to bariatric surgery. The panel of molecular biomarkers consists of the following biomarkers:

[0012] Table 1: Biomarkers of the panel of the invention.

[0013] CYB5, cytochrome b5, A; SYIIG, gamma-synuclein; HBA, hemoglobin alpha subunit; GPX4, glutathione peroxidase 4; GRP78, glucose-regulated protein 78 kDa; SUCB2, succinate-CoA ligase [GDP-forming] beta subunit, mitochondrial; LRP1, Lipoprotein low-density receptor-related protein 1; QCR1, cytochrome b-c1 complex subunit 1, mitochondrial; SLIRF4, locus of excess protein 4; and AIFM1, apoptosis-inducing factor 1, mitochondrial.

[0014] Likewise, the level of oxidized LDL expression has been determined as a surrogate plasma marker of CYB5, which also allows predicting the response to bariatric surgery.

[0015] Therefore, a first aspect of the invention relates to the in vitro use of the level of expression of CYB5 in a sample of subcutaneous adipose tissue from an obese subject susceptible to undergoing bariatric surgery, isolated prior to the bariatric surgery intervention, as a biomarker to predict or prognose the response to bariatric surgery, where underexpressed levels with respect to the average reference values ​​of a group of obese subjects susceptible to undergoing bariatric surgery is an indicator of belonging to the group of subjects that present a moderate response to bariatric surgery.

[0016] We note that, while there are more or less universally accepted criteria in the medical field for establishing obesity levels based on an individual's FMI values ​​(such as an FMI value of 25 or higher indicates overweight, and an FMI value of 30 or higher indicates obesity), the criteria for deciding whether a subject is eligible for bariatric surgery are not as clear or universal. Within the framework of the present invention, an "obese subject eligible for bariatric surgery" is defined as an individual who may benefit from the effects of bariatric surgery and is therefore a candidate for said surgery.

[0017] The beneficial effects of bariatric surgery range from weight loss and the resulting reduction in BMI to improvements in certain clinical parameters associated with obesity, such as those measuring glycemic control (blood glucose or insulin levels) or lipid metabolism (cholesterol, HDL, and LDL levels). Other comorbidities associated with obesity, such as hypertension, dilated cardiomyopathy, and sleep apnea, may also be improved.

[0018] Thus, medical personnel may consider individuals with a BMI above 30 and comorbidities that can be improved by surgery to be candidates for bariatric surgery. These individuals would therefore be considered candidates for "obese individuals susceptible to bariatric surgery." Preferably, an "obese individual susceptible to bariatric surgery" is one with a BMI above 35 and comorbidities, or one with a BMI above 40 without comorbidities. In the present invention, the "comorbidities associated with obesity" include diabetes, hypertension, dilated cardiomyopathy, and sleep apnea.

[0019] In the present invention, a "good response" is defined as a loss of a subject's Body Mass Index (BMI) of 15 months after the intervention, equal to or greater than 30% compared to their pre-intervention BMI. In the present invention, a "moderate response" is defined as a loss of a subject's Body Mass Index (BMI) of less than 30% compared to their pre-intervention BMI, 15 months after the intervention. This 30% BMI loss is based on the median BMI loss experienced by a group of subjects undergoing bariatric surgery 15 months after the intervention.Therefore, in the present invention, a "good response" is defined as a loss of the subject's Body Mass Index (BMI) at 15 months after the intervention, equal to or greater than the median BMI loss experienced by the group of subjects undergoing bariatric surgery after the same period of time. Thus, a "moderate response" is defined as a loss of the subject's Body Mass Index (BMI) at 15 months after the intervention, less than the median BMI loss experienced by the group of subjects undergoing bariatric surgery after the same period of time.In a preferred embodiment of this aspect of the invention, this biomarker, the expression level of CYB5, is used in combination with the expression level of SYIIG, HBA, GPX4, GRP78, SUCB2, LRP1, QCR1, SURF4 and AIFM1, all of them measured in a sample of subcutaneous adipose tissue of subjects with obesity isolated prior to the bariatric surgery intervention, generating a panel of biomarkers to predict or prognose the response to bariatric surgery in subjects with obesity, where underexpressed levels of CYB5, SUCB2, QCR1, SURF4, GRP78, GPX4, AIFM1 and HBA, and overexpressed levels of LRP1 and SYIIG with respect to the average values ​​of an individual with obesity who presents a good response to bariatric surgery are indicators of belonging to the group of subjects who present a moderate response to bariatric surgery.

[0020] Another aspect of the invention relates to the in vitro use of the level of expression of oxidized LDL in an isolated sample of blood, serum or plasma from obese subjects, this being a surrogate marker of CYB5, as a biomarker to predict or prognose the response to bariatric surgery in obese subjects, where overexpressed levels with respect to the average values ​​of an obese individual who presents a good response to bariatric surgery is an indicator of belonging to the group of subjects who present a moderate response to bariatric surgery.

[0021] In the context of the present invention, CYB5 is defined as the amino acid molecules that correspond to the NCBI reference sequences:

[0022] NP_683725.1 - Isoform 1, with sequence SEQ ID NO:1: MAEQSDEAVKYYTLEEIQKH NHSKSTWLILHHKVYDLTKFLEEHPGGEEVLREQAGGDATENFEDVGHSTDAREMSKTF IIGELHPDDRPKLNKPPETLITTIDSSSSWWTNWVIPAISAVAVALMYRLYMAED,

[0023] NP_001905.1 - Isoform 2, with sequence SEQ ID NO:2: MAEQSDEAVKYYTLE EIQKHNHSKSTWLILHHKVYDLTKFLEEHPGGEEVLREQAGGDATENFEDVGHSTDARE MSKTFIIGELHPDDRPKLNKPPEP, o

[0024] NP_001177736.1- Isoform 3, with sequence SEQ ID NO:3: MAEQSDEAVKYYTLEEI QKHNHSKSTWLILHHKVYDLTKFLEEHPGGEEVLREQAGGDATENFEDVGHSTDAREM SKTFIIGELHPETLITTIDSSPASSWWWWLMWWLMW.

[0025] In the context of the present invention, SYUG is defined as the amino acid molecules that correspond to the reference sequence NCBI: NP_003078.2 of sequence SEQ ID NO:4: MDVFKKGFSIAKEGVVGAVEKTKQGVTEAAEKTKEG VMYVGAKTKENVVQSVTSVAEKTKEQANAVSEAVVSSVNTVATKTVEEAENIAVTSGVV RKEDLRPSAPQQEGEASKEKEEVAEEAQSGGD. In the context of the present invention, HBA is defined as the amino acid molecules that correspond to the reference sequence NCBI: NP_000508.1 of sequence SEQ ID NO:5: MVLSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFPT TKTYFPHFDLSHGSAQVKGHGKKVADALTNAVAHVDDMPNALSALSDLHAHKLRVDPVN FKLLSHCLLVTLAAHLPAEFTPAVHASLDKFLASVSTVLTSKYR.

[0026] In the context of the present invention, GPX4 is defined as the amino acid molecules corresponding to the reference sequences:

[0027] NCBI: NP_002076.2 - Isoform A, of sequence SEQ ID NO:6: MSLGRLCRLLKP ALLCGALAAPGLAGTMCASRDDWRCARSMHEFSAKDIDGHMVNLDKYRGFVCIVTNVA FSKICVNGDDAHPLWKWMKIQPKGKGILGNAIKWNFTKFLIDKNGCVVKRYGPMEEPLVI EKDLPHYF,

[0028] NCBI: NP_001034936.1- Isoform B, of sequence SEQ ID NO:7: MSLGRLCRLLKP ALLCGALAAPGLAGTMCASRDDWRCARSMHEFSAKDIDGHMVNLDKYRGFVCIVTNVA SQUGKTEVNYTQLVDLHARYAECGLRILAFPCNQFGKQEPGSNEEIKEFAAGYNVKFDM FSKICVNGDDAHPLWKWMKIQPKGKGILGNAIKWNFTKFGHRLSTVPHRQERLRGEALR THGGAPGDREGPAPLFLAPQGGAFPARAPAHVCGFHRHS,

[0029] NCBI: NP_001034937.1- Isoform C, of ​​sequence SEQ ID NO:8: MGRAGAGSPG RRRQRCQSRGRRRPRAPRRRKAPACRRRRRRRKCPPRSLRPEIHECPKSQDPCA SRDDWRCARSMHEFSAKDIGHMVNLDKYRGFASQTVQQLVQL RYAECGLRILAFPCNQFGKQEPGSNEEIKEFAAGYNVKFDMFSKICVNGDDAHPLWKW

[0030] MKIQPKGKGILGNAIKWNFTKFLIDKNGCWKRYGPMEEPLVIEKDLPHYF, o

[0031] NCBI: NP_001354761.1- Isoform D, of sequence SEQ ID NO:9: MCASRDDWR CARSMHEFSAKDIDGHMVNLDKYRGFVCIVTNVASQUGKTEVNYTQLVDLHARYAECGL RILAFPCNQFGKQEPGSNEEIKEFAAGYNVKFDMFSKICVNGDDAHPLWKWMKIQPKGK GILGNAIKWNFTKFLIDKNGCVVKRYGPMEEPLVIEKDLPHYF.

[0032] In the context of the present invention, GRP78 is defined as the amino acid molecules corresponding to the NCBI reference sequence: NP_005338.1 of sequence SEQ ID NO:10: MKLSLVAAMLLLLSAARAEEEDKKEDVGTVVGIDLGTTYS CVGVFKNGRVEIIANDQGNRITPSYVAFTPEGERLIGDAAKNQLTSNPENTVFDAKRLIGR TWNDPSVQQDIKFLPFKVWEKTKPYIQVDIGGGQTKTFAPEEISAMVLTKMKETAEAYL GKKVTHAVVTVPAYFNDAQRQATKDAGTIAGLNVMRIINEPTAAAIAYGLDKREGEKNILV

[0033] FDLGGGTFDVSLLTIDNGVFEWATNGDTHLGGEDFDQRVMEHFIKLYKKTGKDVRKD NRAVQKLRREVEKKRALSQHQARIEIESFYEGEDFSETLTRAKFEELNMDLFRSTMKP VQKVLEDSDLKKSDIDEIVLVGGSTRIPKIQQLVKEFFNGKEPSRGINPDEAVAYGAAVQA

[0034] GVLSGDQDTGDLVLLDVCPLTLGIETVGGVMTKLIPRNTWPTKKSQIFSTASDNQPTVTI KVYEGERPLTKDNHLLGTFDLTGIPPAPRGVPQIEVTFEIDVNGILRVTAEDKGTGNKNKIT ITNDQNRLTPEEIERMVNDAEKFAEEDKKKERIDTRNELESYAYSLKNQIGDKEKLGGKL SSEDKE™ EKAVEEKI EWLESHQDADI EDFKAKKKLEEI VQPI ISKLYGSAGPPPTGEED TAEKDEL.

[0035] In the context of the present invention, SUCB2 is defined as the amino acid molecules corresponding to the following reference sequences:

[0036] NCBI: NP_001171070.1 de secuencia SEQ ID NO:11 : MASPVAAQAGKLLR ALALRPRFLAGSQAVQLTSRRWLNLQEYQSKKLMSDNGVRVQRFFVADTANEALEAA KRLNAKEIVLKAQILAGGRGKGVFNSGLKGGVHLTKDPNVVGQLAQQMIGYNLATKQTPK EGVKVNKVMVAEALDISRETYLAILMDRSCNGPVLVGSPQGGVDIEEVAASNPELIFKEQI DIFEGIKDSQQRMAENLGFVGPLKSQAADQITKLYNLFLKIDATQVEVNPFGETPEGQV VCFDAKINFDDNAEFRQKDIFAMDKSENEPIENEAAKYDLKYIGLDGNIACVNGAGLA MATCDIIFLNGGKPANFLDLGGGVKEAQVYQAFKLLTADPKVEAILVNIFGGIVNCAIIANGI TKACRELELKVPLVVRLEGTFMEKGSYMHIKQETGNSNENITGIQENVAHQNLSKCAISI FLC, o

[0037] NCBI: NP_003839.2 Isoform 2, of sequence SEQ ID NO:12: MASPVAAQ AGKLLRALALRPRFLAAGSQALTSRRWLNLQEYQSKKLMSDNGVRVQRFFVADTAN EALEAAKRLNAKEIVLKAQILAGGRGKGVFNSGLKGGVHLTKDPNVVGQLAKQMIGYNLA TKQTPKEGVKVNKVMVAEALDISRETYLAILMDRSCNGPVLVGSPQGGVDIEEVAASNPE LIFKEQIDIFEGIKDSQARMAENLGFVGPLKSQAADQITKLYNLFLKIDATQVEVNPFGET

[0038] PEGQWCFDAKINFDDNAEFRQKDIFAMDDXENEPIENEAAKYDLKYIGLDGNIACFVN GAGLAMATCDIIFLNGGKPANFLDLGGGVKEAQVYQAFKLLTADPKVEAILVNIFGGIVNC Al I ANGITKACRELELKVPLVLEGTN VQEAQALPAKKLIKKISA LKVASKIV.

[0039] In the context of the present invention, LRP1 is defined as the amino acid molecules that correspond to the NCBI reference sequence: NP_002323.2 of sequence SEQ ID NO:13: MLTPPLLLLLPLLSALVAAAIDAPKTCSPKQFACRDQITC ISKGWRCDGERDCPDGSDEAPEICPQSKAQRCQPNEHNCLGTELCVPMSRLCNGVQD CMDGSDEGPHCRELQGNCSRLGCQHHCVPTLDGPTCYCNSSFQLQADGKTCKDFDEC SVYGTCSQLCTNTDGSFICGCVEGYLLQPDNRSCKAKNEPVDRPPVLLIANSQNILATYL SGAQVSTITPTSTRQTTAMDFSYANETVCWVHVGDSAAQTQLKCARMPGLKGFVDEHTI NISLSLHHVEQMAIDWLTGNFYFVDDIDDRIFVCNRNGDTCVTLLDLELYNPKGIALDPAM GKVFFTDYGQIPKVERCDMDGQNRTKLVDSKIVFPHGITLDLVSRLVYWADAYLDYIEVV DYEGKGRQTIIQGILIEHLYGLTVFENYLYATNSDNANAQQKTSVIRVNRFNSTEYQVVTR VDKGGALHIYHQRRQPRVRSHACENDQYGKPGGCSDICLLANSHKARTCRCRSGFSLG SDGKSCKKPEHELFLVYGKGRPGIIRGMDMGAKVPDEHMIPIENLMNPRALDFHAETGFI YFADTTSYLIGRQKIDGTERETILKDGIHNVEGVAVDWMGDNLYWTDDGPKKTISVARLE KAAQTRKTLIEGKMTHPRAIVVDPLNGWMYWTDWEEDPKDSRRGRLERAWMDGSHRD IFVTSKTVLWPNGLSLDIPAGRLYWVDAFYDRIETILLNGTDRKIVYEGPELNHAFGLCHHGNYLFWTEYRSGSVYRLERGVGGAPPTVTLLRSERPPIFEIRMYDAQQVGTNKCRV NNGGCSSLCLATPGSRQCACAEDQVLDADGVTCLANPSYVPPPQCQPGEFACANSRCI QERWKCDGDNDCLDNSDEAPALCHQHTCPSDRFKCENNRCIPNRWLCDGDNDCGNS EDESNATCSARTCPPNQFSCASGRCIPISWTCDLDDDCGDRSDESASCAYPTCFPLTQF TCNNGRCININWRCDNDNDCGDNSDEAGCSHSCSSTQFKCNSGRCIPEHWTCDGDND CGDYSDETHANCTNQATRPPGGCHTDEFQCRLDGLCIPLRWRCDGDTDCMDSSDEKS CEGVTHVCDPSVKFGCKDSARCISKAWVCDGDNDCEDNSDEENCESLACRPPSHPCA NNTSVCLPPDKLCDGNDDCGDGSDEGELCDQCSLNNGGCSHNCSVAPGEGIVCSCPL GMELGPDNHTCQIQSYCAKHLKSQKCDQNKFSVKCSCYEGWVLEPDGESCRSLDPFK PFIIFSNRHEIRRIDLHKGDYSVLVPGLRNTIALDFHLSQSALYWTDVVEDKIYRGKLLDNG ALTSFEVVIQYGLATPEGLAVDWIAGNIYWVESNLDQIEVAKLDGTLRTTLLAGDIEHPRAI ALDPRDGILFWTDWDASLPRIEAASMSGAGRRTVHRETGSGGWPNGLTVDYLEKRILWI DARSDAIYSARYDGSGHMEVLRGHEFLSHPFAVTLYGGEVYWTDWRTNTLAKANKWTG HNVTVVQRTNTQPFDLQVYHPSRQPMAPNPCEANGGQGPCSHLCLINYNRTVSCACPH LMKLHKDNTTCYEFKKFLLYARQMEIRGVDLDAPYYNYIISFTVPDIDNVTVLDYDAREQR VYWSDVRTQAIKRAFINGTGVETVVSADLPNAHGLAVDWVSRNLFWTSYDTNKKQINVA

[0040] RLDGSFKNAWQGLEQPHGLVVHPLRGKLYWTDGDNISMANMDGSNRTLLFSGQKGPV GLAIDFPESKLYWISSGNHTINRCNLDGSGLEVIDAMRSQLGKATALAIMGDKLWWADQV SEKMGTCSKADGSGSWLRNSTTLVMHMKVYDESIQLDHKGTNPCSVNNGDCSQLCLP TSETTRSCMCTAGYSLRSGQQACEGVGSFLLYSVHEGIRGIPLDPNDKSDALVPVSGTS LAVGIDFHAENDTIYWVDMGLSTISRAKRDQTWREDVVTNGIGRVEGIAVDWIAGNIYWT DQGFDVIEVARLNGSFRYVVISQGLDKPRAITVHPEKGYLFWTEWGQYPRIERSRLDGTE RVVLVNVSISWPNGISVDYQDGKLYWCDARTDKIERIDLETGENREWLSSNNMDMFSV SVFEDFIYWSDRTHANGSIKRGSKDNATDSVPLRTGIGVQLKDIKVFNRDRQKGTNVCAV ANGGCQQLCLYRGRGQRACACAHGMLAEDGASCREYAGYLLYSERTILSIHLSDERNL NAPVQPFEDPEHMKNVIALAFDYRAGTSPGTPNRIFFSDIHFGNIQQINDDGSRRITIVEN VGSVEGLAYHRGWDTLYWTSYTTSTITRHTVDQTRPGAFARETETVITMSGDDHPRAFVLD ECQNLMFWTNWNEQHPSIMRALSGANVLTLIEKDIRTPNGLAIDHRAEKLYFSDATLDKI ERCEYDGSHRYVILKSEPVHPFGLAVYGEHIFWTDWVRRAVQRANKHVGSNMKLLRVDI PQQPMGIIAVANDTNSCELSPCRINNGGCQDLCLLTHQGHVNCSCRGGRILQDDLTCRA VNSSCRAQDEFECANGECINFSLTCDGVPHCKDKSDEKPSYCNSRRCKKTFRQCSNGR

[0041] CVSNMLWCNGADDCGDGSDEIPCNKTACGVGEFRCRDGTCIGNSSRCNQFVDCEDAS

[0042] DEMNCSATDCSSYFRLGVKGVLFQPCERTSLCYAPSWVCDGANDCGDYSDERDCPGV

[0043] KRPRCPLNYFACPSGRCIPMSWTCDKEDDCEHGEDETHCNKFCSEAQFECQNHRCISK

[0044] QWLCDGSDDCGDGSDEAAHCEGKTCGPSSFSCPGTHVCVPERWLCDGDKDCADGAD

[0045] ESIAAGCLYNSTCDDREFMCQNRQCIPKHFVCDHDRDCADGSDESPECEYPTCGPSEF

[0046] RCANGRCLSSRQWECDGENDCHDQSDEAPKNPHCTSQEHKCNASSQFLCSSGRCVA

[0047] EALLCNGQDDCGDSSDERGCHINECLSRKLSGCSQDCEDLKIGFKCRCRPGFRLKDDG

[0048] RTCADVDECSTTFPCSQRCINTHGSYKCLCVEGYAPRGGDPHSCKAVTDEEPFLIFANR

[0049] YYLRKLNLDGSNYTLLKQGLNNAVALDFDYREQMIYWTDVTTQGSMIRRMHLNGSNVQV

[0050] LHRTGLSNPDGLAVDWVGGNLYWCDKGRDTIEVSKLNGAYRTVLVSSGLREPRALVVD

[0051] VQNGYLYWTDWGDHSLIGRIGMDGSSRSVIVDTKITWPNGLTLDYVTERIYWADAREDYI

[0052] EFASLDGSNRHVVLSQDIPHIFALTLFEDYVYWTDWETKSINRAHKTTGTNKTLLISTLHR

[0053] PMDLHVFHALRQPDVPNHPCKVNNGGCSNLCLLSPGGGHKCACPTNFYLGSDGRTCV

[0054] SNCTASQFVCKNDKCIPFWWKCDTEDDCGDHSDEPPDCPEFKCRPGQFQCSTGICTNP

[0055] AFICDGDNDCQDNSDEANCDIHVCLPSQFKCTNTNRCIPGIFRCNGQDNCGDGEDERD

[0056] CPEVTCAPNQFQCSITKRCIPRVWVCDRDNDCVDGSDEPANCTQMTCGVDEFRCKDS

[0057] GRCIPARWKCDGEDDCGDGSDEPKEECDERTCEPYQFRCKNNRCVPGRWQCDYDND

[0058] CGDNSDEESCTPRPCSESEFSCANGRCIAGRWKCDGDHDCADGSDEKDCTPRCDMD

[0059] QFQCKSGHCIPLRWRCDADADCMDGSDEEACGTGVRTCPLDEFQCNNTLCKPLAWKC

[0060] DGEDDCGDNSDENPEECARFVCPPNRPFRCKNDRVCLWIGRQCDGTDNCGDGTDEED

[0061] CEPPTAHTTHCKDKKEFLCRNQRCLSSSLRCNMFDDCGDGSDEEDCSIDPKLTSCATNA

[0062] SICGDEARCVRTEKAAYCACRSGFHTVPGQPGCQDINECLRFGTCSQLCNNTKGGHLC

[0063] SCARNFMKTHNTCKAEGSEYQVLYIADDNEIRSLFPGHPHSAYEQAFQGDESVRIDAMD

[0064] VHVKAGRVYWTNWHTGTISYRSLPPAAPPTTSNRHRRQIDRGVTHLNISGLKMPRGIAID

[0065] WVAGNVYWTDSGRDVIEVAQMKGENRKTLISGMIDEPHAIVVDPLRGTMYWSDWGNHP

[0066] KIETAAMDGTLRETLVQDNIQWPTGLAVDYHNERLYWADAKLSVIGSIRLNGTDPIVAADS

[0067] KRGLSHPFSIDVFEDYIYGVTYINNRVFKIHKFGHSPLVNLTGGLSHASDVVLYHQHKQPE

[0068] VTNPCDRKKCEWLCLLSPSGPVCTCPNGKRLDNGTCVPVPSPTPPPDAPRPGTCNLQC

[0069] FNGGSCFLNARRQPKCRCQPRYTGDKCELDQCWEHCRNGGTCAASPSGMPTCRCPT

[0070] GFTGPKCTQQVCAGYCANNSTCTVNQGNQPQCRCLPGFLGDRCQYRQCSGYCENFG

[0071] TCQMAADGSRQCRCTAYFEGSRCEVNKCSRCLEGACWNKQSGDVTCNCTDGRVAPS

[0072] CLTCVGHCSNGGSCTMNSKMMPECQCPPHMTGPRCEEHVFSQQQPGHIASILIPLLLLL

[0073] LLVLVAGVVFWYKRRVQGAKGFQHQRMTNGAMNVEIGNPTYKMYEGGEPDDVGGLLD

[0074] ADFALDPDKPTNFTNPVYATLYMGGHGSRHSLASTDEKRELLGRGPEDEIGDPLA. In the context of the present invention, QCR1 is defined as the amino acid molecules that correspond to the NCBI reference sequence: NP_003356.2 of sequence SEQ ID NO: 14: MAASVVCRAATAGAQVLLRARRSPALLRTPALRSTA TFAQALQFVPETQVSLLDNGLRVASEQSSQPTCTVGVWIDVGSRFETEKNNGAGYFLEH LAFKGTKNRPGSALEKEVESMGAHLNAYSTREHTAYYIKALSKDLPKAVELLGDIVQNCS LEDSQIEKERDVILREMQENDASMRDVVFNYLHATAFQGTPLAQAVEGPSENVRKLSRA DLTEYLSTHYKAPRM VLAAAGGVEHQQLLDLAQKH LGGI PWTYAEDAVPTLTPCRFTGS EIRHRDDALPFAHVAIAVEGPGWASPDNVALQVANAIIGHYDCTYGGGVHLSSPLASGAV ANKLCQSFQTFSICYAETGLLGAHFVCDRMKIDDMMFVLQGQWMRLCTSATESEVARG KNILRNALVSHLDGTTPVCEDIGRSLLTYGRRIPLAEWESRIAEVDASVVREICSKYIYDQC PAVAGYGPIEQLPDYNRIRSGMFWLRF.

[0075] In the context of the present invention, SURF4 is defined as the amino acid molecules that correspond to the reference sequences

[0076] <h2 style=";text-align:left;direction:ltr">NCBI: NP_001267717.1 - Isoform 2, de secuencia SEQ ID NO: 15: MFLRVTKQYLPHVA RLCLISTFLEDGIRMWFQWSEQRDYIDTTWNCGYLLASSFVFLNLLGQLTGCVLVLSRNF VQYACFGLFGIIALQTIAYSILWDLKFLMRNLALGGGLLLLLAESRSEGKSMFAGVPTMRE SSPKQYMQLGGRVLLVLMFMTLLHFDASFFSIVQNIVGTALMILVAIGFKTKLAALTLVVWL FAINVYFNAFWTIPVYKPMHDFLKYDFFQTMSVIGGLLLVVALGPGGVSMDEKKKEW,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0077] <h2 style=";text-align:left;direction:ltr"> NCBI: NP_001267718.1 - Isoform 3, de secuencia SEQ ID NO:16: MGQNDLMGTA EDFADQFLRVTKQYLPHVARLCLISTFLEDGIRMWFQWSEQRDYIDTTWNCGYLLASSF VFLNLLGQLTGCVLVLSRNFVQYACFGLFGIIALQTIAYSILWDLKFLMRNLALGGGLLLLL AESRSEGKSMFAGVPTMRESSPKQYLPG,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0078] <h2 style=";text-align:left;direction:ltr"> NCBI: NP_001267719.1 - Isoform 4, de secuencia SEQ ID NO:17: MWFQWSEQRD YIDTTWNCGYLLASSFVFLNLLGQLTGCVLVLSRNFVQYACFGLFGIIALQTIAYSILWDLK FLMRNLALGGGLLLLLAESRSEGKSMFAGVPTMRESSPKQYMQLGGRVLLVLMFMTLLH FDASFFSIVQNIVGTALMILVAIGFKTKLAALTLVVWLFAINVYFNAFWTIPVYKPMHDFLKY DFFQTMSVIGGLLLVVALGPGGVSMDEKKKEW, o<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0079] NCBI: NP_001267721.1 - Isoforma 5, de secuencia SEQ ID NO:18: MGQNDLMGTA EDFADQFLRVTKQYLPHVARLCLISTFLEDGIRMWFQWSEQRDYIDTTWNCGYLLASSF VFLNLLGQLTGCVLVLSRNFVQYACFGLFGIIALQTIAYSILWDLKFLMRLSRTSWAQL.

[0080] In the context of the present invention, AIFM1 is defined as the amino acid molecules that correspond to the NCBI reference sequence: NP_001124318.2 de secuencia SEQ ID NO:19: MGKILPEYLSNWTMEKVRREGVKVMPNAIVQSVGVSSGKL LIKLKDGRKVETDHIVAAVGLEPNVELAKTGGLEIDSDFGGFRVNAELQARSNIWVAGDA ACFYDIKLGRRRVEHHDHAVVSGRLAGENMTGAAKPYWHQSMFWSDLGPDVGYEAIGL VDSSLPTVGVFAKATAQDNPKSATEQSGTGIRSESETESEASEITIPPSTPAVPQAPVQG EDYGKGVIFYLRDKWVGIVLWNIFNRMPIARKIIKDGEQHEDLNEVAKLFNIHED.

[0081] In the context of the present invention, LDL is defined as Low Density Lipoprotein, the main cholesterol transporter in plasma and is composed of approximately proteins (22%), phospholipids (22%), cholesterol (8%), cholesterol esters (42%) and triglycerides (6%). The main LDL protein is apolipoprotein B-100 (apoB-100), a 550 kDa protein that accounts for more than 95% of the total protein mass of LDL, with smaller amounts of other LDL-associated apolipoproteins such as apoE, apoC-III, apoC-II, apoA-I, apoA-IV, apoM, apoJ, serum amyloid A4 (SAA4), calgranulin A, lysozyme C, apoD, apoH, α1-antitrypsin, orosomucoid-1, paraoxonase-1, retinol-binding protein, and prenylcysteine ​​lyase-1 having been described.

[0082] In the context of the present invention, oxidized LDL is defined as a wide range of LDL preparations that have been isolated from biological sources and that have in common the generation or presence of an oxidized particle. There are many in vivo mechanisms capable of oxidizing LDL, including transition metals (iron and copper), and different enzymatic systems such as lipoxygenases, ceruloplasmin, myeloperoxidase, NADPH oxidase and nitric oxide synthase, and it can even be oxidized intracellularly in the lysosomes of macrophages.

[0083] Methods of the invention

[0084] Another aspect of the invention relates to an in vitro method for obtaining data useful for predicting or prognosticating the response to bariatric surgery in obese subjects, prior to the bariatric surgery intervention, hereinafter "first method of the invention" from a biological sample isolated from subcutaneous adipose tissue, comprising: a) quantifying the expression levels of CYB5 and b) comparing the quantities obtained in step (a) with a reference quantity of a group of obese subjects susceptible to undergoing bariatric surgery.

[0085] Preferably, the first method of the invention further comprises quantifying in step a) the expression levels of SYUG, HBA, GPX4, GRP78, SUCB2, LRP1, QCR1, SURF4 and AIFM1 and in step b) comparing the amounts of these biomarkers obtained in step (a) with a reference amount of a group of obese subjects susceptible to undergoing bariatric surgery. In another preferred embodiment, steps (a) and / or (b) may be fully or partially automated.

[0086] Another aspect of the invention relates to an in vitro method for predicting or prognosticating the response to bariatric surgery in obese subjects, prior to the bariatric surgery intervention, hereinafter “second method of the invention”, comprising steps (a) - (b) according to the first method of the invention, and further comprising: c) assigning the individual who presents in an analysis the level of CYB5 expression underexpressed with respect to the average reference values ​​of a group of obese subjects susceptible to undergoing bariatric surgery, to the group of subjects who will present a moderate response to bariatric surgery.

[0087] Preferably, the “second method of the invention” further comprises in step c) assigning the individual who presents in an analysis underexpressed levels of CYB5, SUCB2, QCR1, SURF4, GRP78, GPX4, AIFM1 and HBA, and overexpressed levels of LRP1 and SYIIG with respect to the average reference values ​​of a group of subjects with obesity susceptible to undergoing bariatric surgery to the group of subjects who will present a moderate response to bariatric surgery.

[0088] Another aspect of the invention relates to an in vitro method for obtaining data useful for predicting or prognosticating the response to bariatric surgery in obese subjects, prior to the bariatric surgery intervention, hereinafter "third method of the invention" from a biological sample isolated from blood, serum or plasma, comprising a) quantifying the levels of oxidized LDL expression and b) comparing the quantities obtained in step (a) with a reference quantity of a group of obese subjects susceptible to undergoing bariatric surgery.

[0089] Another aspect of the invention relates to an in vitro method for predicting or prognosticating the response to bariatric surgery in obese subjects, prior to the bariatric surgery intervention, hereinafter "fourth method of the invention", comprising steps (a) - (b) according to the third method of the invention, and further comprising c) assigning the individual who presents in an analysis the level of expression of oxidized LDL overexpressed with respect to the average reference values ​​of a group of obese subjects susceptible to undergoing bariatric surgery, to the group of subjects that will present a moderate response to bariatric surgery. The methods of the invention involve comparing the expression levels of biomarkers in isolated samples from subjects, with the expression levels of said biomarkers in a reference sample or with a median value.

[0090] In the context of the present invention, "reference sample" refers to the sample used to determine the variation in the expression levels of the biomarkers of the present invention. Depending on the biomarker selected, the reference sample will be taken from isolated samples of subcutaneous adipose tissue or from blood, serum, or plasma.

[0091] "Reference value" is the expression level of a biomarker of the invention in a reference sample. "Reference levels" can be determined by measuring expression levels, and those reference levels can be tailored to specific populations; for example, a reference level can be age-related, so that comparisons can be made between expression levels in samples from subjects of a certain age and reference levels for a particular disease, phenotype, or lack thereof, in a certain age group. In a preferred embodiment, the reference value is derived from expression values ​​obtained from a sample of obese individuals amenable to bariatric surgery.Preferably, reference samples are taken from several individuals eligible for bariatric surgery and combined such that the reference value reflects the average value of said biomarkers in the population of individuals eligible for bariatric surgery as previously defined herein. The expression profile in the reference sample may be generated from a population of 2, 3, 4, 5, 10, 15, 20, 30, 40, 50 or more individuals.

[0092] The detection of expression levels of CYB5, SYUG, HBA, GPX4, GRP78, SUCB2, LRP1, QCR1, SURF4, AIFM1, and oxidized LDL can be performed by any means known in the art. The expression levels will give a specific profile. The term "expression level," also called "product quantity" or "expression product quantity," refers to the biochemical material.

[0093] The measurement of the quantity or concentration of the expression product, preferably in a semi-quantitative or quantitative manner, can be carried out directly or indirectly. Direct measurement refers to the measurement of the quantity or concentration of the expression product, which is directly correlated with the number of protein molecules. Such a signal (which we can also refer to as an intensity signal) can be obtained, for example, by measuring an intensity value of a chemical or physical property of said products. Indirect measurement includes measurements obtained from a secondary component or a biological measurement system (e.g., the measurement of cellular responses, ligands, "tags," or enzymatic reaction products).

[0094] The term "quantity," as used in the description, refers to, but is not limited to, the absolute or relative quantity of the expression products, as well as any other values ​​or parameters related to them or that can be derived from them. Such values ​​or parameters include signal intensity values ​​obtained from any of the physical or chemical properties of said expression products obtained by direct measurement. Additionally, such values ​​or parameters include all those obtained by indirect measurement, for example, any of the measurement systems described elsewhere in this document.

[0095] In another preferred embodiment of this aspect of the invention, the result, that is, the expression level of CYB5, SYUG, HBA, GPX4, GRP78, SUCB2, LRP1, QCR1, SURF4 and AIFM1, can be obtained by any method known in the state of the art and specifically any of the following techniques: microarrays, Western blot, mass spectrometry or immunoassays such as ELISA, immunoblot, linear immunoassay (LIA), radioimmunoassay (RIA), immunofluoresence, x-map or protein chips. For immunoassays, "marker compounds" can be used, which as used in the present description, refers to a compound capable of giving rise to a chromogenic, fluorogenic, radioactive and / or chemiluminescent signal that allows the detection and quantification of the amount of antibodies against the proteins of interest.The marker compound is selected from the list comprising radioisotopes, enzymes, fluorophores or any molecule capable of being conjugated to another molecule or detected and / or quantified directly. This marker compound can be bound to the antibody directly, or through another compound. Some examples of marker compounds that are directly bound include, but are not limited to, enzymes such as alkaline phosphatase or peroxidase, radioactive isotopes such as 32P or 35S, fluorochromes such as fluorescein or metallic particles, for direct detection by colorimetry, autoradiography, fluometry or metallography respectively.

[0096] In the method of the present invention, protein expression can be normalized, preferably, in relation to the expression of another protein. Normalization methods are well known in the state of the art. To detect and quantify oxidized LDL, there are various techniques in the state of the art, among which the ELISA technique using two monoclonal antibodies for different epitopes against Apo B, quantification by means of a competitive ELISA, detection by means of immunofluorescence in cell monolayers, spectrophotometric measurement at 232 nm where the absorbance at 232 nm is compared with the absorbance of native LDL.

[0097] The term "comparison" as used in the description refers to, but is not limited to, the comparison of the amount of the expression products of CYB5, SYUG, HBA, GPX4, GRP78, SUCB2, LRP1, QCR1, SURF4, AIFM1 and oxidized LDL of the biological sample to be analyzed, also called the problem biological sample, with a desirable amount of the expression products of CYB5, SYUG, HBA, GPX4, GRP78, SUCB2, LRP1, QCR1, SURF4, AIFM1 and oxidized LDL of one or more reference samples. The reference sample can be analyzed, for example, simultaneously or consecutively, together with the problem biological sample.

[0098] Once the expression levels relative to the reference values ​​have been determined, it is necessary to identify whether there are alterations in expression (increase or decrease in expression). Expression (and levels of the gene expression product) is considered increased in a sample of the subject matter when the levels of increase with respect to the reference sample are at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, 140%, at least 150%, or more.Similarly, expression is considered decreased when its levels decrease relative to the reference sample by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%), at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% (i.e., absent).

[0099] The calculation of said comparison in the methods of the present invention can be performed manually or computer-assisted. The method of the present invention can be applied to samples from individuals of either sex, i.e., men or women, and at any age.

[0100] The invention provides a method for assigning a human subject into one of two groups: group 2, comprising subjects identifiable by the method of the invention, and group 2, representing the remaining subjects.

[0101] It is possible to provide a personalized therapy to an individual depending on whether the individual is assigned to group 1 or group 2. Group 1 comprises subjects identifiable by the method of the invention as individuals who exhibit moderate response to bariatric surgery and group 2 represents the remaining subjects.

[0102] Kit or composition of the invention and uses

[0103] Another aspect of the invention relates to a kit or device, hereinafter referred to as the kit or device of the invention, comprising the elements necessary to quantify the expression level of CYB5. Preferably, it comprises the elements necessary to simultaneously quantify CYB5, SYUG, HBA, GPX4, GRP78, SUCB2, LRP1, QCR1, SURF4, and AIFM1.

[0104] Another aspect of the invention relates to a kit or device, comprising the necessary elements to quantify oxidized LDL. In a preferred embodiment, the kit or device of the invention comprises: (a) means for detecting in a biological sample obtained from the subject the expression levels of the biomarkers of the invention, (b) means for comparing the expression level of the biomarkers determined in (a) with a reference sample, and (c) instructions for a medical professional to prescribe bariatric surgery only to those subjects who can be classified in the good response group.

[0105] The kit or device of the invention may be used, and its use is not particularly limited, although its use in the method of the invention in any of its embodiments is preferred. The kit or device of the invention may comprise controls, program instructions, and information necessary to carry out any of the methods of the invention.

[0106] Automation of the method of the invention by implementing it in a computer program Another aspect of the invention relates to a computer program comprising instructions for performing the procedure according to any of the methods of the invention.

[0107] In particular, the invention encompasses computer programs arranged on or within a carrier. The carrier may be any entity or device capable of supporting the program. When the program is embodied in a signal that can be transported directly by a cable or other device or medium, the carrier may be constituted by said cable or other device or medium. As a carrier, the carrier could be an integrated circuit on which the program is embedded and which has been adapted to execute, or to be used in the execution of, the corresponding processes.

[0108] For example, programs could be embedded in a storage medium, such as a ROM, CD-ROM, or semiconductor ROM, a USB flash drive, or a magnetic recording medium, such as a floppy disk or hard disk. Alternatively, programs could be supported by a transmissible carrier signal; for example, this could be an electrical or optical signal that could be transported via electrical or optical cable, by radio, or by any other means.

[0109] The invention also extends to computer programs adapted to any processing medium capable of carrying out the methods of the invention. Such programs may be in the form of source code, object code, intermediate source code, and object code, for example, in partially compiled form, or in any other form suitable for use in carrying out the processes according to the invention. Computer programs also encompass cloud applications based on such a method.

[0110] Other aspects of the invention relate to the readable storage medium and the transmissible signal comprising program instructions necessary for the execution of the method of the invention by a computer.

[0111] Thus, another aspect of the invention relates to a computer-readable storage medium comprising program instructions capable of causing a computer to perform the steps of any of the methods of the invention.

[0112] Throughout the description and claims, the word "comprise" and its variants are not intended to exclude other technical features, additives, components, or steps. For those skilled in the art, other objects, advantages, and features of the invention will be apparent in part from the description and in part from the practice of the invention. The following examples and drawings are provided for illustrative purposes only and are not intended to limit the present invention.

[0113] BRIEF DESCRIPTION OF THE FIGURES

[0114] Figure 1: Multivariate analysis of proteomic data from study subjects. (A) Highlighted proteins obtained using volcano plot. Overexpressed proteins are shown in red, and downexpressed proteins are shown in blue in the moderate vs. good response group. (B) Pairwise score plots of principal components obtained using PCA. The variances of each PC are shown in each diagonal box. (C) Score plots of the top 2 PCs obtained using PCA. Variances are shown in parentheses. (D) Score plot of the top 2 PCs obtained using PLS-DA. Variances are shown in parentheses. (E) VI P score of differentially expressed proteins in subcutaneous adipose tissue as potential biomarker candidates. 10 proteins. A=Good response; B=Moderate response to bariatric surgery.Red: overexpression, Blue: underexpression.

[0115] Figure 2: Analysis of potential predictive response markers for bariatric surgery. (A) ROC curve for individual proteins obtained by VIP scoring using the classical univariate method. (B) ROC curve for proteins obtained by VIP scoring jointly using the multivariate method. (C) Average of the probabilities and (D) precision value (0.976) performed in 100 cross-validations.

[0116] Figure 3: Scatter plot with the levels of CYB5 expression in the group of subjects with a good response to bariatric surgery (represented in red) and in the group with a moderate response (represented in green).

[0117] Figure 4: Correlation studies: negative correlation between plasma levels of oxidized LDL and CYB5 protein expression prior to bariatric surgery.

[0118] DETAILED DESCRIPTION OF THE INVENTION

[0119] Proteome analysis of subcutaneous adipose tissue in morbidly obese subjects undergoing bariatric surgery to identify predictive response markers for weight loss. This study involved a cohort of obese subjects undergoing bariatric surgery. Subjects (n=12) were monitored before (PRE) and after (POST) the procedure. They were recruited from the Surgery Unit of the Virgen del Rocío University Hospital (Seville). All subjects signed informed consent, which was approved by the relevant Ethics Committee.

[0120] The inclusion criteria for the study were age between 18 and 65 years and BMI >40 kg / m2, or BMI >35 kg / m2 and comorbidities compromised by the presence of obesity itself, such as diabetes, hypertension, dilated cardiomyopathy or sleep apnea.

[0121] Exclusion criteria were the existence of a serious psychiatric disorder, pregnancy, chronic or acute inflammatory disease, oncological disease, asthma, or a history of alcohol and / or drug abuse.

[0122] Anthropometric and biochemical parameters of the subjects were analyzed before and 16 weeks after the intervention and biopsies of subcutaneous adipose tissue were performed at these times to perform a quantitative proteomic study (LC-MS / MS + SWATH) in order to analyze the state of said tissue and to identify possible proteins that would allow discriminating the response to bariatric surgery.

[0123] Subject response was also analyzed 15 months after surgery to establish endpoints for dividing subjects into good responders and moderate responders. The criterion for improved response was the loss of body mass index after surgery, as this is the most standardized indicator of response to bariatric surgery. A good response to bariatric surgery was defined as a decrease in body mass index (BMI) greater than the median BMI loss of the subjects who underwent bariatric surgery at 15 months after surgery. In this group of subjects, the median BMI loss relative to baseline was 31.31%. Two groups of subjects were thus established: a good response with a BMI loss of >30%, and a moderate response with a BMI loss of <30%.

[0124] Characterization of the study subjects before and after bariatric surgery intervention

[0125] Subjects showed an improvement in key anthropometric and metabolic parameters after the intervention (Table 2). Table 2: Anthropometric and biochemical parameters of the study subjects before and after bariatric surgery.

[0126] Values ​​represent the mean ± standard deviation for each parameter. P value obtained using the paired t test. BC, bariatric surgery; BMI, body mass index; WHR, waist-to-hip ratio; HOMA, homeostasis model assessment; HbA1c, glycated hemoglobin; HDL, high-density lipoprotein; LDL, low-density lipoprotein; TNFR2s, serum levels of soluble TNF-α receptor 2; CRP, C-reactive protein. BMI and total fat mass decreased significantly (-34.1% and -47.9%, respectively). Parameters related to carbohydrate metabolism (glucose, insulin, HOMA, HbA1c) also showed significant improvement, with a mean decrease of 23.1%, 66%, 75%, and 14.5%, respectively. Furthermore, 66% of subjects with diabetes experienced remission of their disease. Improvements in lipid profile were less pronounced, with HDL increasing by 28% and triglycerides decreasing by 34.4%.However, neither total cholesterol nor LDL cholesterol, which is an important cardiovascular risk factor, showed a significant improvement in their levels.

[0127] SWATH proteomic analysis of subcutaneous adipose tissue

[0128] A quantitative proteomic study of the subcutaneous adipose tissue of 12 individuals from the initial cohort of obese subjects was performed both before (PRE) and after (POST) undergoing bariatric surgery, using the SWATH methodology (sequential window acquisition of all theoretical fragment ion spectra mass spectrometry).

[0129] Biopsy isolation and protein extraction

[0130] Adipose tissue biopsies were obtained from subjects before and after bariatric surgery, washed in saline solution, and immediately frozen at -80°C until use.

[0131] Identification and quantification of the

[0132] The biopsies were subsequently processed for protein extraction. To do this, the samples (50 mg of tissue) were homogenized in 200 microliters of lysis buffer using a Teflon homogenizer and subsequently sonicated. Finally, the samples were centrifuged, yielding the fat-free aqueous fraction, which was quantified and adjusted for subsequent digestion with trypsin for identification by liquid chromatography-tandem mass spectrometry (LC-MS / MS) and the SWATH (sequential window acquisition of all theoretical fragment ion spectra mass spectrometry) technique. This SWATH technique uses a data-independent MS / MS acquisition method to generate consistently complete, highly specific fragment ion maps that can be queried to determine the presence and quantify any protein of interest, utilizing a targeted data analysis strategy.

[0133] Bioinformatics analysis

[0134] To analyze the proteins and classify them according to their ontological categories (Gen Ontology), the Panther Classification System web platform (http: / / pantherdb.org / ) was used. To carry out the study of predictive markers, the MetaboAnalyst platform (https: / / www.metaboanalyst.ca) was used, a free web platform dedicated to the statistical, functional, and integrative analysis of omics data for the search for biomarkers. Correlation studies

[0135] Linear correlation studies were performed between the expression of proteins of interest and the available anthropometric and biochemical parameters using the IBM SPSS Statistics 25 statistical analysis software.

[0136] Identification of biomarkers

[0137] A proteomic analysis was performed to identify potential preoperative markers of response. A statistical analysis was conducted using the MetaboAnalyst platform to identify potential biomarkers. A first exploratory analysis identified a total of 930 proteins in the adipose tissue proteome, of which 156 showed significant changes in expression after bariatric surgery.

[0138] The MetaboAnalyst web platform was used to identify predictive biomarkers of response to bariatric surgery. A good response to bariatric surgery was defined as a decrease in body mass index (BMI) greater than the median of the subject cohort (31.31%) at 15 months postoperatively. Based on these criteria, our study subjects were divided into two groups: those with a good response to bariatric surgery and those with a poor response to bariatric surgery.

[0139] The volcano plot (Figure 1A) shows the most relevant proteins (fold change threshold / t-test threshold >0.1). The discriminant analysis (Figure 1D) (Partial Least Squares - Discriminant Analysis (PLS-DA)) showed a clear separation of the groups and a top 10 of the best components determined by the Variable Importance in Projection (VIP) value (Figure 1E), which estimates the importance of each variable in the projection used in a PLS (partial least squares) regression model.

[0140] First, a univariate analysis of differential protein expression between the two study groups was performed. This method provides a preliminary view of potential proteins of interest. A fold change (FC) threshold of 2 was set for selecting proteins of interest. For precise selection of potential biomarkers, the volcano plot technique (Figure 1A) was used, which allows the correlation of protein FC with their significance value, obtained using Student's t tests. A p-value of 0.1 was selected as the threshold. Forty-two proteins exceeded this threshold. Among these, CYB5 (log2(FC)=1.5824; Iog10(p)=3.6626) was particularly notable, as it was the protein that presented the best combination of magnitude and significance values ​​for the change in expression between groups.

[0141] Next, a principal components analysis (PCA) was performed. This technique helps group a large number of correlated variables into a significantly smaller number of variables, determining the so-called scores. These scores are reordered to reduce variability, forming the so-called principal components (PCs). The first PC would contain the greatest amount of the sample's variability possible, with the remainder being distributed among the successive PCs. Figure 1B shows the most important PCs in our study along with their variance, which represents the contribution each made to the separation between groups.PCA allows us to identify differences between groups and which subjects contributed most to that difference, which is extremely important for developing predictive models. Figure 1C shows the existence of two groups: group A, in green, which represents the group of subjects who responded well to bariatric surgery, and group B, in red, which represents the subjects with a suboptimal response to said surgery. These graphs reveal the existence of two partially differentiated groups. To enhance this differentiation, techniques such as partial least squares discriminant analysis (PLS-DA) were used. This supervised technique consists of combining the PCs obtained through PCA with multivariate regression models specifically designed to maximize the covariance between the groups established in the study design, as shown in Figure 1D.The main contribution of the PLS-DA technique to this biomarker search study was the creation of a list of highlighted proteins based on a VI P score. Its objective was, by calculating the projection of a given variable within its respective component, to determine those proteins whose presence would best predict the group to which a given subject would belong.

[0142] As observed in Figure 1 E, the protein that registered the highest VI P score was CYB5, coinciding with the result obtained in the univariate analysis.

[0143] These results confirmed CYB5 as the key protein to consider as a potential biomarker. To further explore this protein's potential as a potential marker, the next step was to develop and analyze univariate receiver operating characteristic (ROC) curves, which allow the assessment of a biomarker's prognostic accuracy. They consist of a graphical representation of the relationship between sensitivity and the complementary factor of specificity. They allow the calculation of an index called the area under the curve (AUC), which is a good measure of a biomarker's overall validity.

[0144] The top 10 VI P score candidates were subjected to this analysis. As shown in Figure 2A, the proteins with the best predictive capacity were SYIIG and CYB5 (AUC=1), while the rest of the candidates had an AUC>0.7, indicating a good discriminatory capacity for all of them.

[0145] This analysis was completed with a multivariate ROC curve analysis including the 10 possible candidates (Figure 2B). In this case, an ROC curve of 1 was observed, demonstrating good discriminatory ability between the different groups, as seen in the average of the predicted class probabilities for each sample across the 100 cross-validations (Figure 2C). Furthermore, an accuracy value of 0.976 was determined (Figure 2D).

[0146] The application of this panel of 10 proteins provides high discriminatory power, allowing for accurate and effective prediction of subjects' responsiveness prior to bariatric surgery.

[0147] These results show that CYB5 is a clear candidate biomarker. The CYB5 protein, or cytochrome b5, is an oxidoreductase protein located in the mitochondrial outer membrane whose function is electron transfer within the electron transport chain. Its expression in both groups is shown in Figure 3.

[0148] On the other hand, the panel of molecular biomarkers of response to predictive bariatric surgery, consisting of the 10 top VIP score biomarkers, demonstrates the role of the initial state of adipose tissue as key to successful improvement.

[0149] It is therefore concluded that the prior state of the subjects' subcutaneous adipose tissue could be fundamental in the response derived from bariatric surgery, and could be a parameter to be evaluated in clinical practice. However, since these proteins should be measured in adipose tissue, obtaining the sample would require an invasive method. Therefore, a correlation study with anthropometric and / or plasma parameters was performed in order to reduce invasiveness and identify a surrogate biomarker of the expression of these proteins that is more easily measured. In this sense, it was observed that plasma oxidized LDL values ​​were significantly inversely correlated with the peak intensity levels of CYB5 in the subcutaneous adipose tissue proteome (r = - 0.618; p = 0.043) (Figure 4).

[0150] Thus, oxidized LDL, which is a lipoprotein that is related to the appearance of oxidative stress, could be indicative of the state of adipose tissue prior to the intervention and therefore be a possible predictive marker of response to bariatric surgery.

[0151] The CYB5 protein stands out as a predictive biomarker of response to bariatric surgery, which also correlates with plasma levels of oxidized LDL, which can be used as a surrogate marker of response to bariatric surgery in blood samples.

Claims

CLAIMS 1. In vitro use of the level of CYB5 expression in a sample of subcutaneous adipose tissue from an obese subject susceptible to bariatric surgery, isolated prior to the bariatric surgery intervention, as a biomarker to predict or prognose the response to bariatric surgery, where underexpressed levels with respect to the average reference values ​​of a group of obese subjects susceptible to bariatric surgery is an indicator of belonging to the group of subjects who present a moderate response to bariatric surgery.

2. The in vitro use of the expression level of CYB5 according to the preceding claim, characterized in that it is used in combination with the expression level of SYUG, HBA, GPX4, GRP78, SUCB2, LRP1, QCR1, SURF4 and AIFM1, all of them measured in a sample of subcutaneous adipose tissue from a subject with obesity, isolated prior to the bariatric surgery intervention, where underexpressed levels of CYB5, SUCB2, QCR1, SURF4, GRP78, GPX4, AIFM1 and HBA, and overexpressed levels of LRP1 and SYUG with respect to the average reference values ​​of a group of subjects with obesity susceptible to undergoing bariatric surgery are indicators of belonging to the group of subjects that present a moderate response to bariatric surgery.

3. In vitro use of the level of oxidized LDL expression in an isolated sample of blood, serum or plasma from a subject with obesity as a surrogate marker biomarker of CYB5, to predict or forecast the response to bariatric surgery, where overexpressed levels with respect to the average reference values ​​of a group of subjects with obesity susceptible to undergoing bariatric surgery is an indicator of belonging to the group of subjects who present a moderate response to bariatric surgery.

4. An in vitro method for obtaining data useful for predicting or prognosticating the response to bariatric surgery in obese subjects, prior to the bariatric surgery intervention, from an isolated biological sample of subcutaneous adipose tissue, comprising: a) quantifying the expression levels of CYB5. b) comparing the quantities obtained in step (a) with a reference quantity from a group of obese subjects susceptible to undergoing bariatric surgery.

5. The in vitro method for obtaining useful data according to the preceding claim, which further comprises quantifying in step a) the expression levels of SYUG, HBA, GPX4, GRP78, SUCB2, LRP1, QCR1, SURF4 and AIFM1 and in step b) comparing the quantities obtained in step (a) of these biomarkers with a reference quantity of a group of obese subjects susceptible to undergoing bariatric surgery 6. An in vitro method for predicting or prognosticating the response to bariatric surgery in obese subjects, prior to the bariatric surgery intervention, comprising steps (a) - (b) of claim 4, and further comprising: c) assigning the individual who presents in an analysis the level of CYB5 expression underexpressed with respect to the average reference values ​​of a group of obese subjects susceptible to undergoing bariatric surgery, to the group of subjects who will present a moderate response to bariatric surgery.

7. An in vitro method for predicting or prognosticating the response to bariatric surgery in obese subjects, prior to the bariatric surgery intervention, comprising steps (a) - (b) claim 5, and further comprising: c) assigning the individual who presents in an analysis underexpressed levels of CYB5, SUCB2, QCR1, SURF4, GRP78, GPX4, AIFM1 and HBA, and overexpressed levels of LRP1 and SYUG with respect to the average reference values ​​of a group of obese subjects susceptible to undergoing bariatric surgery, to the group of subjects who will present a moderate response to bariatric surgery.

8. An in vitro method for obtaining data useful for predicting or prognosticating the response to bariatric surgery in obese subjects, prior to bariatric surgery, from a biological sample isolated from blood, serum, or plasma, comprising: a) quantifying the levels of oxidized LDL expression. b) comparing the quantities obtained in step (a) with a reference quantity from a group of obese subjects susceptible to bariatric surgery.

9. An in vitro method for predicting or prognosticating the response to bariatric surgery in obese subjects, prior to the bariatric surgery intervention, comprising steps (a) - (b) of claim 8, and further comprising: c) assigning the individual who presents in an analysis the level of oxidized LDL expression overexpressed with respect to the average reference values ​​of a group of obese subjects susceptible to undergoing bariatric surgery, to the group of subjects who will present a moderate response to bariatric surgery.

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