Izivivinyo ezahlobanisa i-methylene blue nokuphefumula kwamaseli

Igazi lomuntu elikhishwe i-fibrin, eligcinwe lifudumele ku-37 °C amahora amathathu, lidla cishe amaphesenti amabili omoyampilo elinawo. Faka inani elincane le-methylene blue ngaphambi kokuligcina kulawo mazinga okushisa, bese lelo gazi lidla cishe amaphesenti angamashumi ayisithupha nanhlanu. Akukho okushintshile ngamaseli. Yisisombululo kuphela esishintshile.

Lelo nani livela ephepheni lango-1928 likaGeorge Harrop no-E. S. Guzman Barron baseJohns Hopkins, futhi liwumsuka wesitatimende esiphindwa kabanzi sokuthi i-methylene blue ithuthukisa ukuphefumula kwamaseli. Leso sitatimende asilona iqiniso elingamanga, kodwa incazelo yaso ilinganiselwe kakhulu kunalokho okuzwakalayo.

Lokho inani elisethebuleni elikukalayo ngempela

Okwakukalwa ekugcineni kwakuwukuthathwa komoyampilo, okwakunqunywa ngokuhlaziywa kwamagesi. UHarrop noBarron bahlunga igazi elalisanda kuthathwa ngoboya bukakotini ku-10 °C ukuze basuse amaseli amhlophe, bagcwalisa isampula ngomoyampilo, balihlukanisa, bafaka udayi engxenyeni eyodwa, base behlaziya zombili izingxenye ngemva kokuzigcina ezingeni lokushisa elibekiwe. Isibalo samaseli amhlophe sasingavamile ukudlula cishe inkulungwane ngemilimitha eyikhiyubhu. Ukudliwa komoyampilo kwabikwa njengengxenye yomoyampilo wokuqala wesampula eyayisinyamalele.

Baphinde bakala i-carbon dioxide, futhi lapho indlela yokukala yaba nenkinga. Igazi elikhishwe i-fibrin lalikhipha kuze kufike kuma-millimole amabili e-carbon dioxide nge-100 mL ngehora lokuqala lokuligcina ezingeni lokushisa elibekiwe, ngenxa yezizathu ezingahlobene nokusabela okwakucwaningwa. Ngakho izilinganiso ze-carbon dioxide ekhishwayo uma iqhathaniswa nomoyampilo odliwayo egazini eliphelele zazingasebenziseki. Amaseli ageziwe kuphela, asesixazululweni sikaLocke, anikeza amanani ayengachazeka, futhi lawo ayesondelene phakathi kuka-0.73 no-0.80, kunodayi noma ungekho.

Lowo mniningwane awuwona nje into encane engenamsebenzi. “Ukuphefumula” kula maphepha kusho ukushintshana kwamagesi ngaphakathi kwesitsha esivaliwe. Umehluko phakathi kokudliwa komoyampilo nokukhiqizwa kwamandla asebenzisekayo uhlolwa ngokwehlukana kokuthi lokho ukudliwa komoyampilo okukutshela khona nokungakutsheli khona nge-ATP.

Udayi wenezela kakhulu lapho amaseli ayephefumula kancane khona

Ama-erythrocyte avuthiwe ezilwane ezincelisayo cishe awanako ukudliwa komoyampilo okulinganisekayo. Ama-erythrocyte ezinyoni anenucleus ayawudla. UHarrop noBarron bahlola zombili izinhlobo, futhi lowo mehluko uyisisekelo sempikiswano yabo.

Kumasampula ezilwane ezincelisayo udayi wandisa ukudliwa komoyampilo izikhathi ezingamashumi amabili kuya kwezingamashumi amahlanu. Egazini lehansi, elinamaseli ayesevele ephefumula, awuzange wandise izinga ngaphezu kokuliphinda kabili. Lapho ukuphefumula kokuqala kwakuphakeme, udayi wawungeza kancane.

Ukwehla okufanayo komphumela kubonakala nangaphakathi kohlobo olulodwa. Isampula legazi lomuntu owayene-polycythemia vera, elalinamaseli angakavuthwa, ladla amaphesenti angu-11.3 omoyampilo walo kungekho dayi, uma kuqhathaniswa namaphesenti angu-2.8 kumaseli ageziwe avela kumnikeli ophile kahle. Udayi walinyusa lafika kuphela kumaphesenti angu-63.4, ukusheshisa okuphindwe kasithupha lapho amaseli avamile asheshisa cishe izikhathi ezingamashumi amathathu. Udayi wasondeza indlela amaseli avuthiwe asebenza ngayo kweyamaseli angakavuthwa, okuyilokho ababhali abathi wawukwenza.

I-cyanide kwakufanele ikumise lokhu

I-cyanide yayaziwa ngokumisa i-oxidation yamaseli. Noma kunjalo, uHarrop noBarron bayifaka kanye nodayi.

Imiphumela yegazi lenkomo imele lokhu kahle. Umoyampilo wehla usuka ku-22.61 waya ku-22.03 ngokwevolumu ngamaphesenti phakathi namahora amathathu kungekho okwengeziwe okufakiwe. Nge-methylene blue wehla waya ku-12.84. Nge-methylene blue ne-potassium cyanide engu-ingxenye eyodwa kweziyizinkulungwane ezinhlanu ze-molar, wehla waya ku-12.74. Umphumela wodayi wawucishe ungathintekile.

Umfanekiso: irekhodi eliqhubekayo lokuphefumula okuncane kwama-erythrocyte omuntu. Umcibisholo ukhomba lapho kufakwa khona i-methylene blue. Ngaphambi kwalokho, umugqa ucishe ube yisicaba; ngemva kwalokho ukhuphuka ngokuqhubekayo. Kuphinde kwashicilelwa kusuka kuHarrop noBarron, Journal of Experimental Medicine 48:207–223 (1928), PMID 19869478.

Isiphetho sabo kwakuwukuthi udayi wawusebenza njengomthwali womoyampilo, “uhlinzeka ngento esinyamalele kuma-erythrocyte avuthiwe angenanucleus ezilwane ezincelisayo.” Eminyakeni emibili kamuva, uMichaelis noSalomon baseRockefeller Institute babeka ukungavumelani ngendlela eyahlala injalo. Umphumela wodayi wawungaba “yisici esenzeka nje sodayi,” noma udayi wawungaba “yisibonelo sento ethile engaziwa ezintweni eziphilayo.” Lokho kukhetha phakathi kwezincazelo ezimbili akukaxazululwa. Okushintshile ukuthi amaseli manje angachazwa ngokunembe kakhulu.

Okukhishwe esibindini kwenza into efanayo

UMichaelis noSalomon babonisa ukuthi izinto ezikhishwe ezithweni ngamanzi abandayo ziveza ukukhuthazwa okufanayo. Okwesibindi segundane kwakunamandla kakhulu, kulandelwa okwezinso, ubende nesende. Okwengqondo, imisipha kanye ne-serum yegazi kwakunomphumela omncane noma kungenawo nhlobo.

Base behlola lokho okukhishiwe uma kukhona i-carbon monoxide. Ukuphefumula okwakubangelwa yilokho okukhishiwe noma udayi akuzange kushintshe engxubeni yamaphesenti angu-98 e-carbon monoxide namaphesenti angu-2 omoyampilo. Ingxube efanayo yamagesi yavimbela ukuphefumula kwemvubelo nokwama-erythrocyte ezinkukhu anenucleus ngamaphesenti acishe abe ngu-60 kuya ku-80, ngokuhambisana nencazelo kaWarburg yento evubelisayo yokuphefumula ezwelayo ku-carbon monoxide. Ngakho-ke into engaziwa esezicutshini yayingeyona i-enzyme kaWarburg.

Izinto eziningana ezazicatshangwa njengezingaba yiyo zakhishwa ekucatshangelweni phakathi nocwaningo. I-glutathione ne-cysteine ezisesimweni se-reduction zazibangela kuphela ukudliwa komoyampilo okwakudingeka ukuze zona ngokwazo zenze i-oxidation. I-coenzyme kaHarden noYoung yakhishwa ngoba imisipha iqukethe inani elikhulu kakhulu layo kodwa yabonisa cishe umphumela omncane kunayo yonke. I-cytochrome kaKeilin, eyayiqanjwe eminyakeni embalwa ngaphambili, yabhekana nokuphikisana okufanayo. Umbono wabo uqobo wokuthi into esebenzayo yayivela kunucleus yeseli bawubiza ngokuthi uwumbono nje.

Umphumela wawunezimo ezazidingeka

Amaseli kwakudingeka abe ephelele. Ukubhidlika kwamaseli egazi abomvu ngenxa yamanzi acwengiwe ngokuhwamuliswa noma ukuwaqandisa ngokuphindaphindiwe kwaqeda ukusabela kudayi nakulokho okukhishiwe, futhi ukubhidlika kwengxenye yawo kwehlisa ukusabela ngokwesilinganiso sayo.

Ushukela nawo wawudingeka. I-glucose, i-fructose ne-sucrose zazisekela ukuphefumula okubangelwa udayi; i-mannitol ayizange. Uma ungekho ushukela, kwakungekho ukuphefumula nhlobo, futhi udayi wawubonakala ulimaza amaseli ngesikhathi saleyo nqubo, ngakho i-glucose eyayifakwa ngemva kwehora elilodwa noma amabili ayizange ikubuyise ukuphefumula. Ababhali basikisela ukuthi udayi wawenza i-oxidation engxenyeni yesakhiwo iseli elaliyidinga.

Ukukhanya kwandisa kakhulu umphumela wodayi, nakuba umphumela wawucacile nasebumnyameni. Abanye odayi abakwazi ukwenza i-reduction baziphatha ngendlela ehlukile: i-toluylene blue yalingana ne-methylene blue noma yayedlula, kuyilapho i-indigo disulfonate cishe ingenzi lutho. Abukho ubudlelwano obucacile obavela phakathi kwamandla e-oxidation-reduction kadayi nobukhulu bomphumela wawo, okuyinto ababhali abayichaza ngokukwazi kukadayi ukudlula kulwelwesi kanye nobuthi bawo.

Umphumela wawulandela ukuvutshelwa, hhayi ukuphefumula

Ucwaningo lukaBarron lwango-1930 lwasuka ekumisweni kwamaseli oketshezini lwaya ezingcezwini zezicubu zezitho ezivamile zamagundane nezimila ezifakelwe.

Ezicutshini ezingenayo i-glycolysis eyenzeka kukhona umoyampilo, i-methylene blue ayenzanga lutho noma yanciphisa kancane ukudliwa komoyampilo: ezinso cishe ngamaphesenti angu-14, kumanyikwe cishe ngamaphesenti angu-14, esibindini cishe ngamaphesenti angu-5. Ezicutshini ezivubela kukhona umoyampilo, yandisa ukudliwa kwawo: kubende cishe ngamaphesenti angu-8, engqondweni cishe ngamaphesenti angu-13. Ukwanda ezimileni kwakusukela cishe kumaphesenti angu-27 ku-carcinoma yebele lomuntu kuye ngaphezu kwamaphesenti angu-80 ku-Rous sarcoma yenkukhu, cishe ngokwesilinganiso se-glycolysis eyenzeka kukhona umoyampilo esicutshini ngasinye.

Umfanekiso: ichashazi ngalinye liwuhlobo olulodwa lwesicubu. I-eksisi evundlile iyi-glycolysis eyenzeka kukhona umoyampilo; i-eksisi eqondile iwukushintsha kokudliwa komoyampilo ngemva kwe-methylene blue. Idatha ivela kuBarron, Journal of Experimental Medicine 52:447–456 (1930), Ithebula 6, PMID 19869777.

Isivivinyo se-cyanide sabe sesiveza umphumela ohlukile ngokuphambene. Lapho ukuphefumula kulezo zicubu ezazingaphenduli ngaphambili kuvinjelwa nge-cyanide futhi ukuvutshelwa kuvela ngokusobala, udayi waphinde wandisa ukudliwa komoyampilo: ezinso cishe ngamaphesenti angu-59, esibindini cishe ngamaphesenti angu-19, kubende cishe ngamaphesenti angu-286, emasendeni cishe ngamaphesenti angu-200.

UBarron waphakamisa ukuthi udayi ungase usebenze njengesivivinyo samandla okuvubela eseli noma esicubu, futhi waphetha ngomcabango: izizukulwane eziningi zezicubu zesimila ezikhuliswa esixazululweni esinodayi zingase ngolunye usuku zenze ukuguqulwa kwezinto ngaphakathi kwesimila kufane unomphela nokwezicubu ezivamile. Lowo kwakuwumbuzo wocwaningo owabhalwa ngo-1930. Akusona isimangalo sokwelapha.

Lokho izivivinyo ezazingeke zikwazi ukukukala

Ngo-1928 nango-1930, “ukuphefumula” kwakusho ukudliwa komoyampilo. I-adenosine triphosphate yabikwa okokuqala ngo-1929, futhi ukuboniswa kokuthi ama-mitochondria ahlukanisiwe enza i-esterification ye-phosphate ngesikhathi edlulisa ama-electron kwenzeka ngo-1949. Awekho amaphepha okuqala e-methylene blue ayengakwazi ukukala ukukhiqizwa kwamandla, futhi awekho athi ayakukala.

Amaseli ayinkinga yesibili. Ama-erythrocyte avuthiwe ezilwane ezincelisayo awanawo ama-mitochondria. Esigabeni se-reticulocyte akhipha ama-mitochondria nama-ribosome ngenqubo ye-autophagy ekhetha izingxenye ezithile, yingakho iseli legazi elibomvu elivuthiwe lisebenza nge-glycolysis futhi lincike endleleni yalo ye-pentose phosphate ikakhulukazi ukuthola i-NADPH kunokuthola i-ATP. Ezivivinyweni zango-1928 kwakungekho uchungechunge lokuthuthwa kwama-electron olwalungase lulungiswe. Noma yini eyayidla umoyampilo yayenza okunye.

Ukuthi kwakuyini lokho okunye manje sekuchazwe kahle ngokwanele. I-methylene blue yamukela ama-electron avela ezintweni zeseli ezihlinzeka amandla e-reduction, bese iba yi-leucomethylene blue, ephinde yenziwe i-oxidation ngomoyampilo wamangqamuzana, kupheleliswe umjikelezo. Kumaseli egazi abomvu omuntu, i-flavin reductase encike ku-NADPH nama-disulfide reductase ahlobene nayo enza isigaba se-reduction. Ucwaningo lwango-1976 olusebenzisa i-glucose efakwe uphawu lwathola ukuthi udayi ukhuthaza indlela ye-hexose monophosphate, ukuthi umphumela lapho ukugxila kuphansi wawuncike ku-oxyhemoglobin, nokuthi ukugxila okuphezulu, okungu-0.1 mM, kwakukhiqiza i-hydrogen peroxide. Udayi osesimweni se-reduction ohlangana nomoyampilo ukhiqiza i-peroxide, hhayi izibopho ze-phosphate.

Kuma-mitochondria isimo siphikiswa kakhulu kunalokho okuvunywa yizifinyezo ezidumile. Olunye ucwaningo luthi udayi uthatha ama-electron endaweni ye-complex I uwadlulisele ku-cytochrome c. Olunye lwathola ukuthi kuma-mitochondria obuchopho begundane awukwazi ukubuyisa ukuphefumula ngemva kokuvinjwa nge-antimycin, futhi lwabeka indawo eyamukela ama-electron endaweni yokubopha i-ubiquinol ye-complex III esikhundleni salokho. Olwesithathu lwathola ukuthi ukugxila okuphansi kwandisa ukudliwa komoyampilo lapho kungakhuthaziwe, kodwa kwashiya ukuphefumula okukhuthazwa yi-ADP kungashintshile. Isitatimende esiqonde kakhulu saleli phuzu ukuthi ukudliwa komoyampilo okubangelwa i-methylene blue akuncikile nhlobo ku-oxidative phosphorylation: kwaqhubeka lapho uchungechunge lokuphefumula luvinjelwe, nasezinhlelweni zokuhlola ezingenalo.

Kukhona nezilinganiso eziqondile zomehluko phakathi kwalezi zinto ezimbili ezikalwayo. Kuma-mitochondria ahlukanisiwe esibindi segundane, ukwanda kokugxila kodayi kwehlisa izilinganiso zokulawulwa kokuphefumula futhi kwabangela ukuvuvukala, okuyinto ababhali abayichaza ngokuthi ukuhlukaniswa kwe-oxidative phosphorylation. Esibindini segundane esasidluliselwa uketshezi kuso, i-methylene blue yandisa ukudliwa komoyampilo kuyilapho isilinganiso se-ATP uma iqhathaniswa ne-ADP sehla.

Amarekhodi ezokwelapha anomyalezo ofanayo. I-methemoglobinemia yelashwa cishe nge-milligram eyodwa kuya kwamabili e-methylene blue ngekhilogremu, efakwa emthanjeni, ngendlela ye-reduction encike ku-NADPH, kanti imithamo emikhulu ibangela sona isimo eselashwayo. Ukubuyekezwa okufanayo kuphawula ukuthi umphumela wokunqanda ubuthi mkhulu kakhulu kuma-erythrocyte aphelele futhi uyancipha lapho kukhona ukubhidlika kwamaseli egazi abomvu, okuwumphumela wango-1930 wokubhidlika kwalawo maseli ophinde uvele ngemva kweminyaka engamashumi ayisikhombisa nantathu esimweni sasesibhedlela. Into enemiphumela ewusizo neyingozi ezinhlangothini eziphambene zomkhawulo womthamo ayiyona into okufanele umthamo wayo ucatshangwe kususelwa esilinganisweni sokudliwa komoyampilo.

Kungani iqoqo lokukhiqiza liyingxenye yale ngxoxo

UHarrop noBarron baqaphela okuthile okufanele kufundwe okwesibili. I-methylene blue ngokwayo iveza ibhande ku-spectroscope phakathi kwamabhande amabili ayizimpawu ze-methemoglobin, “okungabangela kalula ukudideka uma kuhlolwa ngokushesha.” Udayi ungalingisa into efunwayo.

Inkinga efanayo ikhona nangokwekhemikhali. I-phenothiazine ehlobene nayo ekhona njengengxenye encane inendlela yayo yokwenza i-redox, ikhono layo lokudlula kulwelwesi, nobuthi bayo. Isibonelo esisodwa esibhalwe phansi siveza ngokucacile ukuthi kungani lokhu kubalulekile: ocwaningweni lokufakwa kwe-iodine ku-methylene blue, amasampula ayene-Azure B ecishe ibe ngamaphesenti angu-7 kuya ku-8 akhiqiza ikakhulukazi i-Azure B efakwe i-iodine, ngakho ukungcola kunokuba kube yi-methylene blue kwakubusa ukusabela.

Noma ubani osebenzisa i-methylene blue njenge-reagent, elabhorethri, etafuleni lokudaya amasampula, noma ethangini lezinhlanzi, usebenza ngesithako esingacaciswanga ngaphandle uma iqoqo lokukhiqiza selihlaziyiwe ukuze kwaziwe izici zalo. Izinto ezihlobene nayo, ukuhlonzwa kwayo, ukungcola kwezakhi zamakhemikhali, izincibilikisi ezisele, nensalela ngemva kokushiswa kuyingxenye yesivivinyo, hhayi nje amaphepha ahambisana naso. I-Blupreme ithola i-methylene blue yayo kumkhiqizi wemithi futhi ishicilela isitifiketi esiphelele sokuhlaziywa, ihlola zonke izimfuneko ze-USP kunokuhlola izinsimbi ezisindayo kuphela; umhlahlandlela wokufunda isitifiketi sokuhlaziywa kanye nalokho ukuqokwa njengezinga le-USP okukuqinisekisayo nokungakuqinisekisi kuchaza indlela yokuhlola ukuthi idokhumenti iyahambisana yini nesimangalo.

Inani lalilungile

Izivivinyo zango-1928 nezango-1930 zenziwa ngokucophelela, futhi lokho ezakuqaphela okuyinhloko kusamile: udayi okwazi ukwenza i-reduction ungenza iseli eliphefumula kancane kakhulu lidle umoyampilo izikhathi eziningi kunezinga lalo lapho lingakhuthaziwe, ngaphandle kokudlula esigabeni esivinjelwa yi-cyanide.

Iphutha livela kamuva, lapho “ukuphefumula” kufundwa ngencazelo yakho yesimanje. Ukulinganisa ukushintshana kwamagesi kukutshela ukuthi ama-electron afinyelele kumoyampilo. Akukutsheli ukuthi akhona yini asetshenziselwe ukwenza i-ATP, futhi kuseli elingenawo ama-mitochondria umehluko phakathi kwalezi zitatimende ezimbili mkhulu ngendlela ongaba ngayo. Uhlaka olubanzi lwalokho i-methylene blue ekwenzayo kumaseli lubeka lowo mehluko ofanayo esimweni esibanzi, futhi umlando wale nto kusukela kudayi kuya emithini uchaza ukuthi umbala wezindwangu wafika kanjani kulezi zivivinyo kwasekuqaleni.