I-methylene blue ekuhlolweni kwe-G6PD elabhorethri: lokho udayi akwenzayo ngempela

Umfundi ubuka amashubhu amabili ngemva kokuwagcina ezimweni ezifanele ukuze ukusabela kwenzeke. Elinye seliphenduke umbala olindelekile. Elinye alikaphenduki. Umfundi uphetha ngokuthi isiguli sesibili sintula i-glucose-6-phosphate dehydrogenase. Leso siphetho singase sibe yiqiniso, kodwa indlela afinyelele ngayo kuso yeqa umqondo owenza ukuhlolwa kusebenze. Akukho lutho eshubhuni olukala i-enzyme ngokuqondile. Ishubhu likala lokho okukhiqizwa yi-enzyme, lisebenzisa i-methylene blue njengomxhumanisi.

Uma le ndlela engaqondile isicacile, inqubo, amasampula okulawula nohlu olude lwemiphumela engamanga akusabonakali kuyizinto ezikhethwe nje ngaphandle kwesizathu.

Impendulo emfushane

I-methylene blue ekuhlolweni kwe-G6PD iyinkomba ye-redox. I-enzyme i-G6PD ikhiqiza i-NADPH, bese izinhlelo ezincike ku-NADPH zinciphisa i-methylene blue eluhlaza okwesibhakabhaka ibe uhlobo lwayo lwe-leuco olungenambala, noma ziyisebenzise ukudlulisa ama-electron. Ukusebenza okuvamile kwe-enzyme kwenza ukuncishiswa kwenzeke kahle. Ukusebenza okuntulayo kwenza ukuncishiswa kuhambe kancane noma kungapheleli. Yilokho kuphela okuveza umphumela.

Izindlela ezimbili ezindala zisebenzisa udayi ofanayo kodwa zihlukile kulokho okubhekwa ekugcineni. Uma uzihlukanisa, ukudideka okuningi kuyaphela.

Ukuhlolwa okubili, udayi owodwa, izimpawu zokugcina ezihlukene

Okokuqala ukuhlolwa okuqondile kokuncishiswa kwe-methylene blue, okwachazwa ngu-Sass nozakwabo kwase kuhlolwa kamuva ngu-Gibbs. Lapha okubhekwa ekugcineni udayi ngokwawo: i-methylene blue eluhlaza okwesibhakabhaka esesimweni se-oxidation iyancishiswa isondele ku-leucomethylene blue engenambala, futhi ijubane lalolo shintsho libonisa amandla okukhiqiza i-NADPH. Iphepha lokuqala lokuhlolwa kokuqala lifakwe ohlwini lwe-PubMed ngaphansi kuka-Sass nozakwabo (1966).

Okwesibili ukuhlolwa kwe-Brewer kokuncishiswa kwe-methemoglobin. I-sodium nitrite iqala ngokuguqula i-hemoglobin nge-oxidation ibe yi-methemoglobin ensundu. I-methylene blue ibe isisebenza njengomxhumanisi wokudluliswa kwama-electron: i-methylene blue encishisiwe idlulisela amandla okunciphisa ku-methemoglobin, iyibuyisele ku-hemoglobin ebomvu. Ngakho uphawu olubonakalayo ekugcineni luwumbala onsundu uma uqhathaniswa nobomvu, hhayi oluhlaza okwesibhakabhaka uma uqhathaniswa nokungabi nambala. Indlela yokuqala yavela ku-Bulletin of the World Health Organization (Brewer nozakwabo, 1960).

Ukubiza ukuhlolwa kwe-Brewer ngokuthi ukuhlolwa kokuphela kombala kadayi akuchazi kahle okwenzeka ngokwamakhemikhali. Udayi ushintshashintsha phakathi kwezimo zawo; yi-hemoglobin eshintsha umbala. Umdwebo ongezansi ulandela zombili lezi zindlela.

Ukusabela okuyisisekelo

I-G6PD isheshisa isinyathelo sokuqala se-oxidation endleleni ye-pentose phosphate:

Glucose-6-phosphate + NADP+ becomes 6-phosphogluconolactone + NADPH + H+

Amaseli abomvu egazi asevuthiwe ancike kakhulu kule ndlela ukuze athole i-NADPH, njengoba kubuyekezwe ku-Blood ngu-Luzzatto nozakwabo (2020). I-NADPH igcina i-glutathione isesimweni esincishisiwe futhi ivikela iseli ekulimaleni okubangelwa yi-oxidation. I-methylene blue ingena kulolu hlelo njengomamukeli wama-electron. I-G6PD eyanele ikhiqiza i-NADPH eyanele, eyenza ukuncishiswa kwenzeke kahle. I-G6PD ephansi inqamula lolu chungechunge kusukela esixhumanisini sokuqala.

Cabanga ngodayi njengenkomba yezinga likaphethiloli, hhayi njengenjini. Ubonisa ukutholakala kwe-NADPH. Noma yini enye eshintsha lokho kutholakala, noma ukufundwa komphumela, inganyakazisa inaliti ngaphandle koshintsho esakhini sofuzo.

Indlela inqubo yohlobo lwe-Brewer esebenza ngayo

Izindlela zokulungisa ama-reagent ziye zahluka phakathi namashumi eminyaka, ngakho incazelo yokufundisa akufanele ithathwe njengenqubo yokuhlola iziguli. Ilabhorethri ngayinye ilandela inqubo yayo yokusebenza ejwayelekile eqinisekisiwe. Kucatshangelwa leso sexwayiso, indlela yakudala yamashubhu isebenza kanje, ngendlela echazwe ngamashubhu amathathu nokugcinwa ezimweni zokusabela amahora angu-3 kulokhu kusetshenziswa kwendlela okushicilelwe:

1. Ishubhu lesiguli: igazi eliphelele kanye ne-glucose, i-sodium nitrite ne-methylene blue. I-nitrite yakha i-methemoglobin, i-glucose inikeza amandla ezinqubweni zamaseli abomvu egazi, kanti udayi uhlinzeka ngomxhumanisi wokudlulisa ama-electron oncike ku-NADPH.
2. Isampula yokulawula esesimweni se-oxidation: igazi kanye ne-nitrite ne-glucose kodwa ngaphandle kwe-methylene blue. Ibonisa umbala wokugcina onsundu we-methemoglobin, oyiphethini yokuntuleka.
3. Isampula yokulawula ye-hemoglobin: igazi kanye namanzi. Ibonisa umbala wokugcina obomvu we-hemoglobin, oyiphethini evamile.
4. Gcina ezimweni zokusabela wonke amashubhu ezingeni lokushisa eliseduze no-37 C, ngokuvamile amahora angu-3, uwaxube ngobumnene.
5. Hlambulula ingxenyana encane ethathwe eshubhuni ngalinye emanzini acwengisiswe ngokuhwamulisa ukuze kuqhekeke amaseli futhi imibala iqhathaniseke ngokucacile.
6. Qhathanisa ishubhu lesiguli namasampula amabili okulawula. Ukubuyela ngasembaleni obomvu kubonisa amandla amakhulu okunciphisa. Ukuhlala ngasembaleni onsundu kusikisela ukuntuleka okuphawulekayo.

Ingxube ethile yakudala yayisebenzisa cishe u-1 mL wegazi nama-reagent anezinga elicishe libe ngu-180 mM we-sodium nitrite, 280 mM we-dextrose kanye no-0.4 mM we-methylene blue. Thatha lezo zinombolo njengesibonelo esivela emibhalweni yesayensi, hhayi njengemigomo esebenza yonke indawo.

Ukufunda umphumela

Ukuhlolwa kokuqala okungakali ngobuningi kuhlukanisa kahle kakhulu ukusebenza okuseduze namaphesenti angu-30 kuya ku-40 okusebenza okuvamile, njengoba kuxoxwa kulokhu kubuyekezwa kwe-Malaria Journal kwezikhala ekutholweni kokuntuleka kwe-G6PD. Ngakho umphumela ojwayelekile wokuhlolwa kokuqala usho ukuthi ukuhlolwa akutholanga ukuntuleka okukhulu. Awuqinisekisi ukuthi ukusebenza kuvamile ngokuphelele.

Kungani ukuhlolwa kokuqala kungadukisa

Ukwanda kwama-reticulocyte ngemva kwe-hemolysis kuyisibonelo esaziwayo somphumela ojwayelekile ongamanga. Amaseli abomvu egazi asemasha nama-reticulocyte aqukethe i-G6PD eningi kunamaseli amadala. Ngesikhathi sesigameko esibukhali se-hemolysis, amaseli amadala kakhulu nantula kakhulu angase abe esecekelekile, kusale amaseli asemasha ukusebenza kwawo okuvela kuvamile ekuhlolweni. Lesi simo sezinto eziphilayo sidukisa nokuhlolwa okukala ngobuningi, hhayi ukuhlolwa ngodayi kuphela.

Ukumpontshelwa igazi muva nje kufihla ukuntuleka ngoba amaseli abomvu egazi omnikeli afaka eyawo i-enzyme kusampula. I-Mayo Clinic Laboratories ibala ukumpontshelwa igazi phakathi kwezimbangela zemiphumela ye-enzyme engathembekile, kanye nokwanda kwama-reticulocyte nokuphazamiseka okubangelwa ama-leukocyte.

Abesifazane abanezinhlobo ezimbili ezihlukene zesakhi sofuzo yibo okunzima kakhulu ukubahlola. Ukuvalwa kokusebenza kwe-X-chromosome okwenzeka ngokungahleliwe kwakha ingxube yamaseli abomvu egazi avamile nantulayo, futhi ukusebenza kwawo kukonke kungaba noma kuphi phakathi kokuntuleka okukhulu nokubonakala kuvamile. Ukuhlolwa kokuqala okungakali ngobuningi kuvame ukungawatholi ama-phenotype aphakathi. Imibhalo ye-WHO ngokuhlolwa kwe-G6PD ichaza lo mkhawulo ngokuningiliziwe.

Isikhathi esidlulile kusukela isampula yathathwa, izinga lokushisa lokuyigcina, ukuncika kombala wokugcina ekuboneni komuntu, i-hematocrit nekhwalithi yama-reagent kwenezela ukuphazamiseka kokuhlaziya phezu kwale miphumela yezinto eziphilayo.

Ukuhlolwa kokuqala akukhona ukuxilonga

Umphumela wokuhlolwa kokuqala obonisa ukuntuleka, ukusebenza okuphakathi noma ongahambisani nesimo sesiguli udinga ukuhlolwa kwe-enzyme i-G6PD okukala ngobuningi. I-WHO ibheka i-spectrophotometry ekala ngobuningi ngezinga lokushisa elilawulwayo njengendlela eyisilinganiso sokuqhathanisa. Kulokho kuhlolwa, ingxube yamaseli abomvu egazi aqhekeziwe ifakwa i-glucose-6-phosphate ne-NADP+, bese i-NADPH eyakhiwayo ilandelwa ngokwanda kokumuncwa kokukhanya ku-340 nm. Ijubane loshintsho lilingana nokusebenza kwe-enzyme, ngokuvamile okuvezwa maqondana ne-hemoglobin njengamayunithi ngegremu ngalinye. Indlela esetshenziswa manje emalabhorethri okuhlola iziguli ifingqiwe encazelweni yokusebenza kwe-G6PD kukhathalogi yokuhlolwa ye-Mayo.

Ukuchazwa kwemiphumela kusebenzisa ibanga eliqinisekiswe yilabhorethri njengelokuqhathanisa, ngokuvamile elichazwa maqondana nokusebenza okuvamile emphakathini, kanti ukuntuleka kuvame ukuchazwa eduze komkhawulo wamaphesenti angu-30 nokusebenza okuphakathi kube ngaphezu kwawo. Amaphrofayili e-WHO ezici ezihlosiwe zemikhiqizo yokuhlola i-G6PD (2022) achaza lolo hlaka lokuxilonga. Uma i-hemolysis noma ukumpontshelwa igazi okwenzeke muva nje kwenza ukuhlonza i-phenotype kungathembeki, phinda ukuhlolwa ngemva kokuba inani nezinhlobo zamaseli abomvu egazi sezibuyele esimweni sokulingana; ukuhlolwa kwamangqamuzana nakho kungenezela ulwazi ngaphandle kokuthatha indawo yokukalwa kwe-enzyme.

Umngcele okufanele ugcinwe

I-methylene blue kulokhu kuhlolwa iyi-reagent yaselabhorethri esetshenziswa ngaphandle komzimba ophilayo. Lelo qiniso liyingxenye yengxoxo ehlukile kweyokuphepha kokunikeza isiguli i-methylene blue, echazwe eziqondisweni ngezimo lapho i-methylene blue kungafanele isetshenziswe khona. I-biochemistry iyazixhumanisa lezi zinto, ngoba nomuthi uncike ekuncishisweni okuncike ku-NADPH, kodwa udayi oseshubhini lokuhlola awuwona umthamo osegazini lomuntu. Ukuze uthole umongo wokuthi lo dayi usebenza kanjani ekubaliseni amasampula kwesinye isimo, bheka izimiso zokubala amasampula nge-methylene blue, kanti mayelana nomndeni wamakhemikhali oyingxenye yawo, bheka odayi be-redox elabhorethri.

Ukuhlolwa kokuqala kubuza ukuthi amandla okunciphisa abonakala ephelele yini. Ukuhlolwa okuqinisekisayo kubuza ukuthi ingakanani i-enzyme ekhona ngempela. Udayi uwuphendula kahle umbuzo wokuqala, uma nje kungekho muntu owucela ukuba uphendule owesibili.