Massa spectermeter arqiliq yaghning hazim qilinish ahwalini olchash va uning yurak qan-tomur kesalliri dora tatqiqatidiki

roliPharmacokinetics, pharmacodynamics and drug metabolism/ Merck and

Company, Rahway, New jerseyMay 05th, 2012

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MazmunlarMazmunlar

Yagh metabolizimi va uning yurak qan tomur kesallikliri dora sahasidki roli

• CVD arqiliq olganlar toghruluq statestika

• Yagh metabolizimi

Yaghing hazim qilininishini olchashta ishlitilidighan massa spectermeterliri

• Quadropole and trap

• Izitop nisbiti Mass Spectermetiri

• Yuquri eniqliq MS

• Ion yotkilishchanlighi mass spectrometeri

Izitop agashkuchisini ishlitish arqiliq yaghning toshulishini va badandin chiqiriwetilishini takshurush

• DNL-Yagh kesilitasi va and cholesterolni sinsizlash

• Yaghing oksidlinip qelishi

3© World Health Organization, Factsheet N*310., updated 2011

Dunyadiki 10 chong olush sawapliri

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A A Simplified Simplified Model of Lipid MetabolismModel of Lipid Metabolism

FC = Free CholFC = Free CholCE = Chol EsterCE = Chol EsterTG = TriglycerideTG = TriglycerideFFA = FFA = Free Fatty AcidFree Fatty AcidFecesFeces Peter TothPeter Toth, , Drugs,Drugs, 2010; 70 (11): 1363-1379 2010; 70 (11): 1363-1379FECES

Gut

Liver

PlaquePlaque

LDLLDL

HDLHDL

ChylosChylos

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Arqa korunush: Yaghlar badanning harqaysi jaylirida Arqa korunush: Yaghlar badanning harqaysi jaylirida yaghahsillirigha (Lipoproteins) qoshulup toshuluduyaghahsillirigha (Lipoproteins) qoshulup toshuludu

“Apolipoproteins” qandiki yaghlarni “lipoproteins” dap atilidighan partikollar arqiliq toshuydu.

• Bu arqiliq suda erimaydighan towandiki yaghlar qanda toshulidu:

– Triglycerides (TGs), Cholesterol (C), Cholesterol Esters, (CE) Phospholipids (PL), Free Fatty Acids (FFA)

– Hydrophobic compounds must also be carried (albumin, lipoproteins)

Lipoproteins• Chylomicrons (CM)

– ApoB48, Yimakliklardin kelidighan, uchayda

• Towan zichliqdiki Lipoprotein (VLDL)

– ApoB100, jigarda boludighan yagh– Yagh hazim bolghandin keyin IDL/LDL gha aylinidu.

• Yuquri zichliqliq Lipoprotein (HDL)

– ApoA1, Badan muskuliridiki yagh.

LDLLDL

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Next topic…Next topic…

Yagh metabolizimi va uning yurak qan tomur kesallikliri dora sahasidki roli

• CVD arqiliq olush san-sepiri

• Yagh metabolismi

Yaghing hazim qilinishini alchashta ishlitilidighan massa spectermeterliri

• Quadropole and trap

• Izitop nisbiti Mass Spectermetiri

• Yuquri eniqliq MS

• Ion yotkilishchanlighi mass spectrometeri

Izitop agashkuchisini ishlitish arqiliq yaghning toshulishini va bandandin chiqiriwetilishini takshurush

• DNL-Yagh kesilitasi va and cholesterolni sinsizlash

• Yaghing oksidlinip qelishi

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Massa spectometerni yagh Massa spectometerni yagh metabolizimini takshrushta ishltish..… metabolizimini takshrushta ishltish..… Masiliga qarap uskuna tallashMasiliga qarap uskuna tallash

me

as

ure

d r

esp

on

se, t

race

r in

corp

ora

tio

n

expected labeling, metabolic rate

0.00

0.25

0.50

0.75

1.00

0.00 0.25 0.50 0.75 1.00

GCGC--IRMSIRMS

GCGC--((q)MSq)MS

cellscells

me

as

ure

d r

esp

on

se, t

race

r in

corp

ora

tio

n

expected labeling, metabolic rate

0.00

0.25

0.50

0.75

1.00

0.00 0.25 0.50 0.75 1.00

GCGC--IRMSIRMS

GCGC--((q)MSq)MS

cellscells

GC & GC-MS/MS

Thermo IRMS

Agilent GC/MS

GCFID

High Res MSThermo LTQ-FTThermo OrbitrapAgilent QToFBruker QqFTICRIon Mobility MSDrift tube IMS Waters Synapt G

Tandem Quads, Ion Traps

Thermo TSQ Cohesive

Thermo TSQ Quantum

Waters Xevo TQ

Thermo LTQ

Waters Quattro Micro

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GC-MS or MS/MS..GC-MS or MS/MS..Single or triple quad possibilitiesSingle or triple quad possibilities

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VentVent

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Fatty acid labeling via a bigger mouse trap?Fatty acid labeling via a bigger mouse trap?

Quadrupole Collision Cell ICR CellSource

ESI (- ion)

3.3 ul/min @ 0.05 mg/ml

4200 capV

Isolate (2 Da window)255.2 – 257.2 m/z

OFF Narrow band acquisition

1 Da window 255.7 – 256.7 m/z

average 50 spectra (4.2 sec/scan)

~ 600,000 resolving @ 256 m/z

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Orthogonal separation by T-wave IM-MS (LC-MSOrthogonal separation by T-wave IM-MS (LC-MSEE) )

Wrona, Mauriala, Bateman, Mortishire-Smith and O'Connor. RCMS, 2005

Castro-Perez, Kamphorst, DeGroot, Lafeber, Goshawk, Yu, Shockcor, Vreeken and Hankemeier. J Proteome Res, 2010

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Biologiyalik maddilarni ayrishBiologiyalik maddilarni ayrish

Parchilangha massilarni va ana massini birla Parchilangha massilarni va ana massini birla waqitta olchiyalaydu.waqitta olchiyalaydu.

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Next topic..Next topic..

Yagh metabolizimi va uning yurak qan tomur kesallikliri dora sahasidki roli

• CVD arqiliq olush san-sepiri

• Yagh metabolismi

Yaghing hazim qilinishini alchashta ishlitilidighan massa spectermeterliri

• Quadropole and trap

• Izitop nisbiti Mass Spectermetiri

• Yuquri eniqliq MS

• Ion yotkilishchanlighi mass spectrometeri

Izitop agashkuchisini ishlitish arqiliq yaghning toshulishini va bandandin chiqiriwetilishini takshurush

• DNL-Yagh kesilitasi va and cholesterolni sinsizlash

• Yaghing oksidlinip qelishi

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Yagh va ahsilning ishlap chiqirili..Yagh va ahsilning ishlap chiqirili..

TriglyceridesTriglycerides

DGDG

Cholesterol EstersCholesterol Esters

PhospholipidsPhospholipids

CH3-CoA-oxidation-oxidation

Yagh kesiltaStored/Dietary Stored/Dietary lipidslipids

2H2O

[2H]-cholesterol [2H]-Palmitate

Ahsil (ApoA and B etc.)

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0.0

0.2

0.4

0.6

0.8

m0 m1 m2 m3 m4 m16

% d

istr

ibu

tio

n%

dis

trib

uti

on

mass isotopomermass isotopomer

Massa izitomerlirini olchash bashlanghuchMassa izitomerlirini olchash bashlanghuch uqumuqum

CHCH33 – (CH – (CH22))1414 - COOH - COOHC16 x 12 = 192C16 x 12 = 192H32 x 1 = 32H32 x 1 = 32O2 x 16 = 32O2 x 16 = 32

256 257 258 259 260 272

MW 256 MW 256

molecular weightmolecular weight

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Orientation towards the figuresOrientation towards the figures

timetime

labelinglabelingm1/m0m1/m0

newly made material newly made material == labeling in the productlabeling in the product

labeling in the precursorlabeling in the precursorx poolx pool

excess labelingexcess labelingm1/m0m1/m0tracertracer – m1/m0 – m1/m0baselinebaseline

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Chol

FA-CoAFA-CoA

MTP+ ApoB

DGlysoPA PA TGCE

FA-CoAMGFatty Acyl-CoA(FA-CoA)

PL

VLDL

MTP+ ApoB

DGlysoPA PA TGCE

FA-CoA

PL

Chol

FA-CoAFA-CoA

[[22H]FAH]FA

Putkul obrazni bir qetimdila tamamlash..Putkul obrazni bir qetimdila tamamlash..

DGAT2

Incorporation of 2H Fatty Acidinto Triglycerides is DECREASED by DGAT2i

FAO?

2H17 Oleic Acid

2H2O

2H2O

[2H]acetyl-CoA

CO2

GC-IRMS

TriglyceridesPhospholipids

Cholesteryl EstersDiglycerides

LC-MSMeasure fat oxidation

Measure lipid changes

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Yaghning oksidlinishini takshurush:Yaghning oksidlinishini takshurush:- Experimental flow scheme- Experimental flow scheme

Oral gavage Oral gavage 22HH-Fatty Acid-Fatty Acid

Predose, 2.5 hrs and 5 hrsPredose, 2.5 hrs and 5 hrs

CollectCollect plasmaplasma 10 uL Plasma10 uL Plasma

2 uL 10N NaOH, 15uL acetone, 2 uL 10N NaOH, 15uL acetone, spin & incubate 4 hrsspin & incubate 4 hrs acetoneacetone

Plasma & NaOHPlasma & NaOH

GC-IRMSGC-IRMS

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Sample in AcetoneSample in Acetone

Reference Reference

Convert raw data intoConvert raw data into percent labelingpercent labeling

0

200

400

600

800

1000

0.000 0.005 0.010 0.015 0.020 0.025expected enrichment (% excess 2H)

HD

: H

H (

del

ta)

GC-IRMS of acetone labelingGC-IRMS of acetone labeling

0

500

1000

1500

2000

2500

3000

3500

0 30 60 90 120 150 180 210 240time (seconds)

60 90 120 150 180 210 24060 90 120 150 180 210 240time (seconds)

sig

nal

in

ten

sity

(m

V)

0

500

1000

1500

2000

2500

3000

3500

0 300

500

1000

1500

2000

2500

3000

3500

0 30

sig

nal

in

ten

sity

(m

V)

Compare HD/HHCompare HD/HH of Reference and Sampleof Reference and Sample

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Olchash usulini sinaqdin otkuzushOlchash usulini sinaqdin otkuzush

Izitopning miqdarining yagh oksidlinishigha tasiri barmu?

Malikulidiki izitop sanining yagh oksidlinishigha tasiri barmu?

Yaghning oksidlinishini tohtidighan dorining suning izitop qoyuqliqigha tasiri barmu?

50 or 150 mg/kg [d31]16:050 or 150 mg/kg [d31]16:0

150 mg/kg of [d17] or [d34]18:1150 mg/kg of [d17] or [d34]18:1

150 mg/kg of [d17]18:1 ± etomoxir150 mg/kg of [d17]18:1 ± etomoxir

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150

250

350

450

550

0 1 2 3 4 5

[2H34]oleate (150 mg x kg-1)

[2H17]oleate (150 mg x kg-1)

[2H31]palmitate (150 mg x kg-1)

[2H31]palmitate (50 mg x kg-1)

0 1 2 3 4 5 0 1 2 3 4 5

control, [2H17]oleate (150 mg x kg-1)

etomoxir, [2H17]oleate (150 mg x kg-1)

time (hours)

2H

-la

be

lin

g o

f w

ate

r(d

elt

a v

s r

efe

ren

ce

ga

s)

Yagh kesilitasining oksidlinishi. Naziriyani

DD22O ning hasil bolushi towandiki amillarning tasiriga uchrighan: O ning hasil bolushi towandiki amillarning tasiriga uchrighan:

(A)(A)Yaghning miqdariYaghning miqdari(B)(B)Yaghning izitop qoyuqlughi Yaghning izitop qoyuqlughi (C)(C)etomoxiretomoxir

AA BB CC

7:00am7:00am 11:00am11:00am 1:30pm1:30pm 4:00pm4:00pm

FastingFasting DosingDosing

0Hr0Hr 2.5Hrs2.5Hrs 5Hrs5Hrs

n = 5-7 per group

7:00am7:00am 11:00am11:00am 1:30pm1:30pm 4:00pm4:00pm

FastingFasting DosingDosing

0Hr0Hr 2.5Hrs2.5Hrs 5Hrs5Hrs

n = 5-7 per group

Naziriyani chachqanda tajriba qilip korush

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AcknowledgementsAcknowledgementsAnalytical Biochemistry Stephen PrevisThomas RoddyJose Castro-PerezKithsiri HerathDavid McLarenYi Pan Rory RohmAnita LeePaul Miller Mark Rosenbach Vinit ShahLani CardasisKatie SouthwickSteven Stout Dan Xie Nykia WalkerWeixun WangHaihong Zhou

AtherosclerosisDoug JohnsSheng Ping WangAlison StrackBrian HubbardCheng Ying

Experimental MedicineIrene NunesAizza Hassan

Clinical Development LabMichael Lassman

ESD OperationsJennifer McCarthy

MBMX/PPDMMichele ClearyLisa Shipley

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Rahmat