頂空氣相(xiang)色(se)譜系(xi)列講座（18）
人體(ti)血液中(zhong)一氧化碳的分(fen)析——分(fen)光光度(du)法定量5
三、分光(guang)光(guang)度法(fa)
1、紫外光譜法原理
CO中(zhong)毒血(xue)(xue)中(zhong)可能含(han)有三(san)種血(xue)(xue)紅(hong)(hong)(hong)(hong)蛋(dan)(dan)(dan)(dan)(dan)(dan)白： ⑴碳(tan)(tan)氧血(xue)(xue)紅(hong)(hong)(hong)(hong)蛋(dan)(dan)(dan)(dan)(dan)(dan)白（HbCO）——與CO結合(he)(he)的(de)Hb； ⑵ 氧合(he)(he)血(xue)(xue)紅(hong)(hong)(hong)(hong)蛋(dan)(dan)(dan)(dan)(dan)(dan)白——與O2結合(he)(he)的(de)Hb ⑶血(xue)(xue)紅(hong)(hong)(hong)(hong)蛋(dan)(dan)(dan)(dan)(dan)(dan)白——沒(mei)結合(he)(he)的(de)Hb。 它們在500nm~600nm范圍內(nei)吸(xi)收(shou)光(guang)譜(pu)見(jian)(jian)圖-2。碳(tan)(tan)氧血(xue)(xue)紅(hong)(hong)(hong)(hong)蛋(dan)(dan)(dan)(dan)(dan)(dan)白zui大吸(xi)收(shou)峰分別(bie)在568.2nm和(he)538nm；氧合(he)(he)血(xue)(xue)紅(hong)(hong)(hong)(hong)蛋(dan)(dan)(dan)(dan)(dan)(dan)白zui大吸(xi)收(shou)峰分別(bie)在576.2nm和(he)542nm；血(xue)(xue)紅(hong)(hong)(hong)(hong)蛋(dan)(dan)(dan)(dan)(dan)(dan)白zui大吸(xi)收(shou)峰在555nm。如(ru)血(xue)(xue)液含(han)三(san)種形式(shi)的(de)血(xue)(xue)紅(hong)(hong)(hong)(hong)蛋(dan)(dan)(dan)(dan)(dan)(dan)白（其中(zhong)的(de)微量(liang)高鐵血(xue)(xue)紅(hong)(hong)(hong)(hong)蛋(dan)(dan)(dan)(dan)(dan)(dan)白可忽略(lve)不計），則其光(guang)譜(pu)呈現三(san)種光(guang)譜(pu)的(de)混合(he)(he)形。如(ru)在血(xue)(xue)液中(zhong)加入還原劑(ji)（連二亞(ya)硫酸鈉或硫化(hua)銨）溶(rong)液，則可將氧合(he)(he)血(xue)(xue)紅(hong)(hong)(hong)(hong)蛋(dan)(dan)(dan)(dan)(dan)(dan)白及(ji)高鐵血(xue)(xue)紅(hong)(hong)(hong)(hong)蛋(dan)(dan)(dan)(dan)(dan)(dan)白還原為血(xue)(xue)紅(hong)(hong)(hong)(hong)蛋(dan)(dan)(dan)(dan)(dan)(dan)白，而碳(tan)(tan)氧血(xue)(xue)紅(hong)(hong)(hong)(hong)蛋(dan)(dan)(dan)(dan)(dan)(dan)白不被還原，因而血(xue)(xue)液中(zhong)只(zhi)存(cun)在碳(tan)(tan)氧血(xue)(xue)紅(hong)(hong)(hong)(hong)蛋(dan)(dan)(dan)(dan)(dan)(dan)白和(he)血(xue)(xue)紅(hong)(hong)(hong)(hong)蛋(dan)(dan)(dan)(dan)(dan)(dan)白兩種不同的(de)形式(shi)，它們的(de)吸(xi)收(shou)光(guang)譜(pu)見(jian)(jian)圖-1
用分(fen)光光度法測定血中(zhong)HbCO定性和(he)(he)定量是(shi)(shi)基于(yu)HbCO和(he)(he)對光譜的(de)(de)特征(zheng)吸(xi)收(shou)不同(tong)，和(he)(he)特征(zheng)吸(xi)收(shou)與濃(nong)度有(you)(you)(you)量的(de)(de)關系。HbO2在(zai)589nm~577nm和(he)(he)556nm~536nm區間有(you)(you)(you)兩條(tiao)吸(xi)收(shou)譜帶，其zui大(da)吸(xi)收(shou)是(shi)(shi)579nm和(he)(he)542nm。HbCO在(zai)579nm~564nm和(he)(he)548nm~530nm區間有(you)(you)(you)兩條(tiao)吸(xi)收(shou)譜帶，其中(zhong)zui大(da)吸(xi)收(shou)是(shi)(shi)572nm和(he)(he)539nm。加入還原劑后檢血中(zhong)HbO2和(he)(he)高(gao)鐵血紅(hong)蛋(dan)白(bai)被(bei)還原為血紅(hong)蛋(dan)白(bai)（Hb），此時吸(xi)收(shou)譜帶變為一(yi)條(tiao)，即在(zai)530nm~580nm之間，zui大(da)吸(xi)收(shou)為556nm。而(er)HbCOzui大(da)吸(xi)收(shou)波長(chang)仍是(shi)(shi)572nm和(he)(he)539nm不變。
2、具體操作步驟
取CO中(zhong)毒心血(xue)（檢材血(xue)）0.1ml~0.3ml，加10ml 0.1%硫化銨(an)或多硫化銨(an)水溶液(ye)（內含連二(er)亞硫酸鈉2.5mg/ml）稀釋混勻，測定(ding)500nm~600nm之間的吸(xi)收(shou)圖(tu)譜(pu)，用切線(xian)法(fa)(fa)量(liang)取555nm和540nm處的吸(xi)收(shou)值見(jian)圖(tu)-2，計(ji)算血(xue)中(zhong)HbCO% = F ×（A540/B555）×100公式(shi)，式(shi)中(zhong)F為(wei)常(chang)數1.5；A540見(jian)圖(tu)-2中(zhong)a~b（cm）線(xian)段長；B555見(jian)圖(tu)-2中(zhong)c~d（cm）線(xian)段長。此(ci)法(fa)(fa)適合檢材血(xue)中(zhong)含量(liang)HbCO% > 40% 時效果好，此(ci)方法(fa)(fa)在辦案中(zhong)使用較(jiao)多。
3、另(ling)一種經(jing)驗方法
適合各種(zhong)CO濃度(du)檢(jian)(jian)材(cai)血的(de)HbCO%測定，用0.4%氨水(shui)溶液稀釋的(de)含CO血在529nm和582nm處(chu)有吸(xi)收(shou)峰(feng)，稀釋液做參比。根據經(jing)驗公(gong)式：HbCO% = （A529－A582）×185/（A529－0.07）進(jin)行計算。185和0.07均為經(jing)驗常數，本(ben)方法要求稀釋檢(jian)(jian)材(cai)血的(de)吸(xi)光度(du)值在0.4~0.6范圍準確度(du)高。
