Gwaje-gwajen da suka danganta methylene blue da numfashin tantanin halitta

Jinin ɗan adam da aka cire masa fibrin, aka dumama shi zuwa 37 °C na tsawon awa uku, yana amfani da kusan kashi biyu cikin ɗari na oxygen da yake ɗauke da shi. Idan aka ƙara ɗan methylene blue kafin ajiye shi a wannan yanayin zafi, wannan jinin yana amfani da kusan kashi sittin da biyar cikin ɗari. Babu abin da ya canja game da ƙwayoyin jinin. Ruwan da suke ciki ne kawai ya canja.

Wannan adadi ya fito ne daga wata takardar bincike ta 1928 da George Harrop da E. S. Guzman Barron suka rubuta a Johns Hopkins, kuma shi ne asalin maganar da ake yawan maimaitawa cewa methylene blue yana inganta numfashin tantanin halitta. Maganar ba ƙarya ba ce, amma ma’anarta ta fi yadda take ji ƙunci.

Abin da adadin da ke cikin jadawalin yake aunawa a zahiri

Abin da aka auna a ƙarshe shi ne shan oxygen, wanda aka tantance ta hanyar nazarin iskar gas. Harrop da Barron sun tace jinin da aka ɗebo sabo ta auduga a 10 °C domin cire fararen ƙwayoyin jini, suka cika samfurin da oxygen, suka raba shi, suka ƙara rini a wani ɓangaren, sannan suka nazarci duka biyun bayan an ajiye su a yanayin zafin gwajin. Adadin fararen ƙwayoyin jini ba kasafai ya wuce kusan dubu ɗaya a kowace milimita mai siffar kubu ba. An bayyana amfani da oxygen a matsayin kaso na oxygen da samfurin yake ɗauke da shi tun farko wanda ya ɓace.

Sun kuma auna carbon dioxide, amma a nan hanyar aunawar ta kawo matsala. Jinin da aka cire masa fibrin ya saki har millimole biyu na carbon dioxide a kowace 100 mL a awa ta farko ta gwajin, saboda wasu dalilai da ba su da alaƙa da abin da ake nazari. Don haka, ba a iya amfani da rabon carbon dioxide da aka saki da oxygen da aka sha daga cikakken jinin ba. Ƙwayoyin jini da aka wanke aka saka cikin ruwan Locke ne kawai suka ba da alkaluman da za a iya fassara, kuma waɗannan sun kasance tsakanin 0.73 da 0.80, ko da rinin yana ciki ko babu shi.

Wannan bayani ba ƙaramin abu ba ne. “Numfashi” a cikin waɗannan takardun yana nufin musayar iskar gas a cikin rufaffiyar mazubi. An bincika bambancin da ke tsakanin amfani da oxygen da samar da kuzari mai amfani dabam a cikin abin da amfani da oxygen yake faɗa da abin da ba ya faɗa game da ATP.

Rinin ya fi ƙara tasiri inda ƙwayoyin jinin suka fi ƙarancin numfashi

Cikakkun jajayen ƙwayoyin jinin dabbobi masu shayarwa kusan ba sa amfani da oxygen gwargwadon abin da za a iya aunawa. Jajayen ƙwayoyin jinin tsuntsaye masu cibiyar tantanin halitta kuwa suna yi. Harrop da Barron sun gwada duka biyun, kuma wannan bambanci shi ne ginshiƙin hujjarsu.

A samfuran dabbobi masu shayarwa, rinin ya haifar da ƙaruwa daga ninki ashirin zuwa ninki hamsin. A jinin goose, wanda ƙwayoyinsa tuni suke numfashi, bai taɓa ƙara saurin fiye da ninki biyu ba. Yayin da numfashin farko ya fi yawa, abin da rinin zai iya ƙarawa ya fi raguwa.

Irin wannan bambancin ya bayyana a cikin nau’in halitta ɗaya. Wani samfurin jinin ɗan adam daga majiyyaci mai polycythemia vera, wanda ya ƙunshi ƙwayoyin jini da ba su gama girma ba, ya yi amfani da kashi 11.3 cikin ɗari na oxygen ɗinsa ba tare da rini ba, idan aka kwatanta da kashi 2.8 cikin ɗari a ƙwayoyin jini da aka wanke daga mai ba da jini mai lafiya. Rini ya kai shi kashi 63.4 cikin ɗari kawai, wato saurin ya ƙaru ninki shida, alhali na ƙwayoyin jini na yau da kullum ya ƙaru kusan ninki talatin. Rinin ya sa cikakkun ƙwayoyin jini suka kusanci halayen waɗanda ba su gama girma ba, kuma haka marubutan suka ce yake yi.

Ya kamata cyanide ya dakatar da shi

An san cyanide yana dakatar da oksidashan a cikin tantanin halitta. Duk da haka, Harrop da Barron sun ƙara shi tare da rinin.

Sakamakon jinin shanu yana wakiltar abin da aka gani. Yawan oxygen ya sauka daga 22.61 zuwa 22.03 a ma’aunin kaso na girman oxygen cikin ɗari na girman jini cikin awa uku ba tare da an ƙara komai ba. Da methylene blue, ya sauka zuwa 12.84. Da methylene blue da potassium cyanide mai ƙarfin molar ɗaya bisa dubu biyar, ya sauka zuwa 12.74. A zahiri, tasirin rinin bai canja ba.

Hoto: bayanan numfashi a ƙaramin ma’auni na jajayen ƙwayoyin jinin ɗan adam da aka rubuta ba tare da yankewa ba. Kibiya tana nuna lokacin da aka ƙara methylene blue. Kafin haka, layin ya kasance kusan madaidaici; bayan haka ya riƙa hawa a hankali ba tare da tsayawa ba. An sake gabatar da shi daga Harrop da Barron, Journal of Experimental Medicine 48:207–223 (1928), PMID 19869478.

Sun kammala da cewa rinin ya yi aiki a matsayin mai ɗaukar oxygen, “yana samar da wani sinadari da ya ɓace daga jajayen ƙwayoyin jinin dabbobi masu shayarwa da suka balaga, waɗanda ba su da cibiyar tantanin halitta.” Bayan shekaru biyu, Michaelis da Salomon a Rockefeller Institute sun bayyana saɓanin ra’ayin ta hanyar da ta dawwama. Ko dai tasirin rinin “wata sifa ce ta rinin da ta faru kwatsam,” ko kuma rinin “samfurin wani sinadari ne da ba a sani ba a cikin halittu.” Har yanzu ba a yanke hukunci tsakanin waɗannan hanyoyi biyu ba. Abin da ya canja shi ne yanzu ana iya bayyana waɗannan ƙwayoyin jini da daidaito sosai.

Wani tsantsar hanta ya yi irin wannan abu

Michaelis da Salomon sun nuna cewa tsantsar gaɓoɓin jiki da aka samu da ruwan sanyi suna haifar da irin wannan ƙaruwa. Hantar bera ta fi tasiri, sai ƙoda, saifa, da ƙwayar halitta. Kwakwalwa, tsoka, da ruwan jini sun yi ɗan tasiri ko ba su yi ba.

Daga nan suka gwada tsantsar da carbon monoxide. Numfashin da tsantsar ko rinin ya haifar bai canja ba a cakuda kashi 98 cikin ɗari na carbon monoxide da kashi 2 cikin ɗari na oxygen. Wannan cakudar iskar gas ta hana numfashin yisti da na jajayen ƙwayoyin jinin kaji masu cibiyar tantanin halitta da kusan kashi 60 zuwa 80 cikin ɗari, daidai da bayanin Warburg game da wani sinadarin enzyme na numfashi da carbon monoxide yake shafa. Saboda haka, sinadarin naman jikin da ba a gano ba ba enzyme na Warburg ba ne.

An yi watsi da wasu sinadarai da ake zargin su ne a yayin binciken. Glutathione da aka yi wa ridakshan da cysteine sun haifar da amfani da oxygen da ake buƙata don oksidashan nasu kawai. An cire coenzyme na Harden da Young daga jerin saboda tsoka ce ta fi ɗauke da shi, amma ta nuna kusan mafi ƙarancin tasiri. Cytochrome na Keilin, wanda aka ba suna wasu shekaru kaɗan kafin hakan, ya ci karo da wannan saɓanin ma. Shawarar da su kansu suka bayar cewa sinadarin mai aiki ya fito daga cibiyar tantanin halitta, sun kira ta shawara kawai.

Tasirin yana da sharuɗɗa

Dole ne ƙwayoyin jinin su kasance cikakku ba tare da fashewa ba. Fashewar jajayen ƙwayoyin jini ta hanyar ruwa da aka tsarkake da tururi ko daskarewa akai-akai ta kawar da martanin da suke yi ga rinin da tsantsar duka, kuma fashewar wani ɓangare na ƙwayoyin jinin ta rage martanin daidai gwargwado.

Ana kuma buƙatar sukari. Glucose, fructose, da sucrose sun goyi bayan numfashin da rinin ya haifar; mannitol bai yi haka ba. Idan babu sukari, babu numfashi ko kaɗan, kuma rinin ya nuna alamun lalata ƙwayoyin jinin a yayin hakan. Don haka, glucose da aka ƙara bayan awa ɗaya ko biyu bai dawo da numfashin ba. Marubutan sun ba da shawarar cewa rinin yana yin oksidashan ga wani ɓangaren tsarin ƙwayar jinin da take buƙata.

Haske ya ƙara tasirin rinin sosai, ko da yake tasirin ya bayyana a cikin duhu. Sauran rinannuka da za a iya yi wa ridakshan sun nuna halaye daban-daban: toluylene blue ya kai ko ya zarce methylene blue, yayin da indigo disulfonate kusan bai yi komai ba. Ba a samu wata alaƙa bayyananniya tsakanin ƙarfin oksidashan-ridakshan na rini da girman tasirinsa ba, abin da marubutan suka danganta da iya ratsa membrane da gubarsa.

Tasirin ya bi fermenteshan, ba numfashi ba

Binciken Barron na 1930 ya tashi daga ƙwayoyin halitta da aka gauraya cikin ruwa zuwa siraran yankan nama daga gaɓoɓin beraye na al’ada da ƙari da aka dasa.

A naman jikin da ba ya yin glycolysis a gaban oxygen, methylene blue bai yi komai ba ko kuma ya ɗan rage amfani da oxygen: a ƙoda da kusan kashi 14 cikin ɗari, a pancreas da kusan kashi 14 cikin ɗari, a hanta da kusan kashi 5 cikin ɗari. A naman jikin da yake yin fermenteshan a gaban oxygen, ya ƙara amfani da shi: a saifa da kusan kashi 8 cikin ɗari, a kwakwalwa da kusan kashi 13 cikin ɗari. Ƙaruwar a ƙari ta kasance daga kusan kashi 27 cikin ɗari a carcinoma na nonon ɗan adam zuwa fiye da kashi 80 cikin ɗari a sarcoma na Rous a kaza, kusan daidai gwargwado da glycolysis a gaban oxygen na kowane naman jikin.

Hoto: kowace ɗigo tana wakiltar nau’in naman jiki guda. Ma’aunin kwance yana nuna glycolysis a gaban oxygen; ma’aunin tsaye yana nuna canjin amfani da oxygen bayan methylene blue. Bayanai daga Barron, Journal of Experimental Medicine 52:447–456 (1930), Jadawali 6, PMID 19869777.

Daga nan gwajin cyanide ya haifar da sakamako akasin na farko. Lokacin da aka toshe numfashi da cyanide a waɗannan naman jikin da a baya ba su nuna martani ba, fermenteshan kuma ya bayyana, rinin ya sake ƙara amfani da oxygen: a ƙoda da kusan kashi 59 cikin ɗari, a hanta da kusan kashi 19 cikin ɗari, a saifa da kusan kashi 286 cikin ɗari, a ƙwayar halitta da kusan kashi 200 cikin ɗari.

Barron ya ba da shawarar cewa za a iya amfani da rinin wajen gwada ƙarfin tantanin halitta ko naman jiki na yin fermenteshan, sannan ya rufe da wani zato: renon ƙari na tsararraki da yawa a matsakaicin reno mai ɗauke da rini zai iya wata rana sa tsarin sarrafa sinadaransa ya zama kamar na naman jiki na al’ada har abada. Wannan tambayar bincike ce da aka rubuta a 1930. Ba da’awar magani ba ce.

Abin da gwaje-gwajen ba za su iya aunawa ba

A 1928 da 1930, “numfashi” yana nufin amfani da oxygen. An fara bayar da rahoton adenosine triphosphate a 1929, kuma sai a 1949 aka nuna cewa mitochondria da aka ware suna haɗa phosphate ta esterification yayin da suke canja wurin electron. Babu ɗaya daga cikin takardun farko kan methylene blue da zai iya auna samar da kuzari, kuma babu ɗayansu da ya yi da’awar yin hakan.

Ƙwayoyin jinin su ne matsala ta biyu. Cikakkun jajayen ƙwayoyin jinin dabbobi masu shayarwa ba su ƙunshi mitochondria ba. A matakin reticulocyte, suna kawar da mitochondria da ribosomes ta hanyar autophagy mai zaɓe. Shi ya sa cikakkiyar jajayen ƙwayar jini take dogara da glycolysis, kuma take amfani da hanyar pentose phosphate galibi don samun NADPH maimakon ATP. A gwaje-gwajen 1928, babu jerin jigilar electron da za a gyara. Duk abin da yake amfani da oxygen yana yin wani abu ne dabam.

Yanzu an bayyana wannan wani abin dabam da kyau sosai. Methylene blue yana karɓar electron daga sinadaran tantanin halitta masu yin ridakshan, ya zama leucomethylene blue, wanda oxygen na ƙwayoyin molecule yake sake yi wa oksidashan, don haka zagayen ya cika. A jajayen ƙwayoyin jinin ɗan adam, flavin reductase mai dogara da NADPH da disulfide reductases masu alaƙa da shi suna aiwatar da matakin ridakshan. Wani bincike na 1976 da ya yi amfani da glucose mai alamar ganowa ya gano cewa rinin yana ƙara aikin hanyar hexose monophosphate, cewa tasirin a ƙaramin matakin taro ya dogara da oxyhemoglobin, kuma cewa matakin taro mafi girma, 0.1 mM, ya samar da hydrogen peroxide. Rinin da aka yi wa ridakshan idan ya haɗu da oxygen yana samar da peroxide, ba ɗaurin phosphate ba.

A cikin mitochondria, batun ya fi samun saɓanin ra’ayi fiye da yadda taƙaitattun bayanan da suka shahara suke nunawa. Wani ɓangaren bincike yana cewa rinin yana ɗaukar electron daga yankin complex I ya kai su ga cytochrome c. Wani kuma ya gano cewa a mitochondria na kwakwalwar linzamin kwamfuta, ba ya iya dawo da numfashi bayan antimycin ya toshe shi, kuma ya sanya wurin karɓar electron a wurin ɗaure ubiquinol na complex III maimakon haka. Na uku ya gano cewa ƙananan matakan taro sun ƙara amfani da oxygen a yanayin hutu, amma ba su canja numfashin da ADP ya motsa ba. Bayanin da ya fi kai tsaye shi ne cewa amfani da oxygen da methylene blue yake haifarwa ba ya dogara da fosforileshan ta oksidashan ko kaɗan: ya ci gaba lokacin da aka hana aikin jerin numfashi, har ma a samfuran da aka shirya waɗanda ba su da wannan jerin.

Akwai kuma ma’aunai kai tsaye na bambancin da ke tsakanin waɗannan abubuwa biyu. A mitochondria na hantar bera da aka ware, ƙara matakin taron rini ya rage rabon sarrafa numfashi kuma ya haifar da kumburi, abin da marubutan suka bayyana a matsayin warewar haɗin fosforileshan ta oksidashan. A hantar bera da aka riƙa wuce ruwa ta cikinta, methylene blue ya ƙara amfani da oxygen yayin da rabon ATP da ADP ya ragu.

Bayanan asibiti suna ɗauke da saƙo iri ɗaya. Ana magance methemoglobinemia da kusan milligram ɗaya zuwa biyu na methylene blue a kowace kilogram ta cikin jijiya, ta hanyar ridakshan mai dogara da NADPH, kuma manyan allurai suna haifar da yanayin da ake magancewa. Wannan bita ta kuma lura cewa tasirin kawar da gubar ya fi ƙarfi a jajayen ƙwayoyin jini da ba su fashe ba, kuma yana raguwa idan akwai fashewar ƙwayoyin jini. Wannan shi ne abin da aka gano game da fashewar ƙwayoyin jini a 1930 yana sake bayyana bayan shekaru saba’in da uku a yanayin asibiti. Sinadarin da tasirinsa mai amfani da mai cutarwa suke a ɓangarori biyu na iyakar adadin allura ba sinadari ne da ya kamata a tantance allurarsa daga ma’aunin amfani da oxygen ba.

Dalilin da ya sa rukunin samarwa yake da muhimmanci a tattaunawar

Harrop da Barron sun lura da wani abu da ya cancanci a sake karantawa. Methylene blue kansa yana haifar da wani layi a na’urar spectroscope tsakanin layuka biyu na musamman na methemoglobin, “wanda zai iya haifar da ruɗani cikin sauƙi idan aka duba ba tare da zurfafa ba.” Rinin zai iya yin kama da sinadarin da ake nema.

Wannan matsalar tana kuma da wani salo na sinadarai. Wani phenothiazine mai alaƙa da shi wanda yake a matsayin ƙaramin ɓangare na samfurin yana da nasa halin oksidashan-ridakshan, nasa ikon ratsa membrane, da nasa guba. Wani lamari da aka rubuta ya nuna muhimmancin batun a zahiri: a binciken haɗa iodine da methylene blue, samfuran da suka ƙunshi kusan kashi 7 zuwa 8 cikin ɗari na Azure B sun samar galibi da Azure B da aka haɗa da iodine. Don haka, ƙazantar ce ta mamaye halayyar sinadaran maimakon methylene blue.

Duk wanda yake amfani da methylene blue a matsayin sinadarin gwaji, a dakin gwaje-gwaje, a teburin sanya launi ga samfura, ko a tankin kifaye, yana aiki da wani ɓangaren haɗi da ba a tantance ba sai idan an tantance rukunin samarwar. Sinadarai masu alaƙa da shi, ainihin sinadarin, ƙazantar abubuwan asali, ragowar sinadaran narkarwa, da abin da ya rage bayan ƙonawa suna cikin gwajin, ba takardun da suke kewaye da shi kawai ba. Blupreme yana samun methylene blue nasa daga masana’antar magunguna kuma yana wallafa cikakkiyar takardar shaidar nazari, yana gwada dukkan ƙa’idojin USP maimakon ƙarafa masu nauyi kawai; jagorar karanta takardar shaidar nazari da abin da alamar matakin USP take tabbatarwa da abin da ba ta tabbatarwa ba suna bayanin yadda za a duba takardar a kwatanta da da’awar.

Adadin ya kasance daidai

An gudanar da gwaje-gwajen 1928 da 1930 da kulawa, kuma babban abin da suka lura da shi ya ci gaba da tabbata: rini da za a iya yi wa ridakshan zai iya sa tantanin halitta da kusan ba ya numfashi ya yi amfani da oxygen sau da yawa fiye da saurin da yake yi a hutu, ba tare da wucewa ta matakin da cyanide yake toshewa ba.

Kuskuren yana bayyana daga baya, lokacin da aka karanta “numfashi” da ma’anarsa ta zamani. Ma’aunin musayar iskar gas yana gaya maka cewa electron sun isa ga oxygen. Ba ya gaya maka cewa an yi amfani da wani daga cikinsu wajen samar da ATP, kuma a tantanin halitta da ba shi da mitochondria, bambancin waɗannan maganganu biyu yana da girma matuƙa. Faɗaɗɗen taswirar abin da methylene blue yake yi a cikin ƙwayoyin halitta tana sanya wannan bambanci a cikin bayani mafi faɗi, kuma tarihin sinadarin daga rini zuwa magani yana bayyana yadda rinin yadi ya fara shiga waɗannan gwaje-gwajen.