general status for nutrition or severe organ disease

단백질 - 혈액성분 중 혈구세포를 제외한 가장 많은 부분을 차지 - general status for nutrition or severe organ disease (protein losing state) - 건강 성인 총단백 : 약 7 g/d. L (혈장의 7%) 총단백(protein) 6. 6 -8. 3 g/d. L 알부민 (albumin) 3. 5 -5. 2 g/d. L 글로불린 (globulin) 총단백-알부민 - 수 백 가지의 개별 단백질들로 이루어짐 - 개별 단백의 농도 증감을 통해 대략적인 신체 상태 파악 (간, 신장, 장, 염증…) 알부민 : more informative (nutritional state, liver synthetic capacity protein losing nephropathy or enteropathy )

Measurements Chemical assay 1) Total protein 2) Albumin 3) Globulins (=total protein – albumin) Protein electrophoresis (PEP) - fractionation (separation) of globulins from albumin (highly specific pattern of major serum proteins for some diseases) - quantitation by scanning of stained protein (0. 1 g/d. L) : not reliable Immunoassay for individual proteins

Protein Detection & Quantitation 1. 표준법 : Kjeldahl method - analysis of nitrogen content (단백의 질소함유율 16%) protein Acid digestion --------> ammonium ion (ammonium nitrogen x 6. 25) 정밀한 방법, 아미노산마다 질소함유량 달라 다양한 단백질 함유한 검체에 부적절 2. 굴절률법 3. 비중법 4. Biuret법 - Colorimetry ① Biuret method : m/c use in automated chemistry analyzer peptide bond + Cu 2+ ------> purple complex ② Lowry protein assay : higher sensitivity, phenol reagent (=Folin-Ciocalteau reagent) Biuret method + phenol reagent (oxidation of aromatic protein residues) 5. 색소결합법 : 혈청내 여러 단백이 특정 색소에 특이적으로 결합하는 성질 이용 Coomassie brilliant blue (CBB), bromphenol blue (BPB), amido black 10 B, Pyrogallol red (urine total protein), Ponceau S…

Technique of Protein Separation 1. 전기영동 (Electrophoresis, EP) 1) 원리 : 전해질 용액에 전류를 가했을 때 용액 중 입자들이 전하량과 용매 사이의 마찰저항에 따른 고유의 이동 속도로 양극 또는 음극으로 이동함으로서 분리 가능 2) EP에 영향을 주는 조건 ① 완충액의 p. H : p. H에 따라 단백 입자의 하전량이 달라짐 ② 전기장 : 전압 ↑ -> 분리시간 & 단백확산 ↓ (heat : 단백변성 및 확산, 증발) ③ 완충액의 이온 강도 : 이온강도 ↑ -> 저항 ↓ ④ 지지체 : agarose, cellulose acetate, polyacrylamide cf) SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) SDS : anionic detergent (denaturation of protein) -> even distribution of charge -> fractionation by MW 3) Result EP, Gel staining, Scanning, Densitometer(밀도계), Electrophoretogram serum PEP -> 알부민 분획 + 글로불린 분획(α 1, α 2, β, γ)

Protein electrophoresis (PEP) pattern in agarose gel Serum protein + - Plasma protein

Technique of Protein Separation 2. Precipitation (침전) sodium sulfate, sodium sulfite, ammonium sulfate, methanol 첨가 -> 글로불린은 침전되고 알부민만 남음 (부정확: α 글로불린) -> original serum & precipitate (or supernatant)에서 각각 단백을 측정 -> A/G ratio 3. Column separation 1) Gel filtration chromatography - size exclusion 2) Ion exchange chromatography 3) Affinity chromatography 4) Capillary electrophoresis 4. 질량분석기

Pattern of Protein Abnormalities No abnormal finding Polyclonal gammopathy

Pattern of Protein Abnormalities Monoclonal gammopathy Nephrotic syndrome Serum Urine 소실양상 : small large (albumin -> α 1 -AT, Tf, … -> Ig) Very large molecules (α 2 -MG) & large micelle of β-LP : retained in serum!

Pattern of Protein Abnormalities Liver cirrhosis (Polyclonal gammopathy) Albumin 생산감소 For maintenance of oncotic pressure : Ig 생산 증가 (polyclonal nature) Ig. A도 증가 : β-γ bridging

Immunofixation electrophoresis (IFE) - 2 techniques : agarose gel protein EP + immunoprecipitation Ag antisera (Ab) -> precipitation of Ag-Ab complex (removal of unreacted proteins) -> staining -> direct comparison of IFE bands with corresponding protein EP pattern - ID of monoclonal protein (M protein, paraprotein) : class & type

IEP (immunoelectrophoresis) - 2 techniques : agarose gel protein EP + immunodiffusion (antisera) - precipitin arc formation : control과 환자 비교 (size, location & shape) - ID of monoclonal protein : class & type (Ig G, A, M, D, E, κ, λ) Dense, bowed Ig. G arc Diminished Ig. A level No Ig. M Bowed, abnormal κ arc

Specific proteins Major components Minor components Readily detected on EP gels Not usually detected by standard EP Low level, immunologic methods Prealbumin, Albumin α 1 At, α 2 MG, Haptoglobin(Hpt) β-LP, Tf, Fibrinogen(Fibr), 보체 Ceruloplasmin(Cer), Gc globulin Hemopexin(Hpx) α 1 -acid glycoprotein(α 1 Ac), CRP

Prealbumin - 전기영동 상에서 알부민보다 더 양극으로 이동하는 단백 - 62 k. Da - Binding to thyroxine (thyroxine binding prealbumin, TBPA or transthyretin, TTR) - Role in metabolism of vitamin A prealbumin-RBP complex prealbumin-RBP-vitamin A(retinol) complex - Marker for nutritional status : rapid dynamics of synthesis & clearance - short half-life (약 2일) - sensitive to intake of adequate nutrition & to alteration in hepatic function : better early indicator than albumin or Tf - albumin or Tf : abundant 다른 요인들에 의한 영향, slower scale - Easy cross into CSF : EP of normal CSF – a major peak of albumin + prealbumin & small Tf

Albumin - Most abundant protein in normal plasma (usually upto 2/3 of total protein) - Liver - Half-life 약 17일, 크기 66, 000 Da - 기능 : Intravascular oncotic pressure Mobile repository of amino acids Transport or carrier protein (thyroxine, bilirubin, cortisol, estrogen, FFA, penicillin, warfarin, Ca, Mg. . ) - 측정 1) Dye binding methods Bromcresol green (BCG) : m/c use in automated chemistry analyzer BCP (purple), BPB… 2) 전기영동에 의한 분획비로부터 환산 3) urine albumin : immunoassay (TIA)

Albumin - Glycated albumin (Glycoalbumin, glycosylated albumin) 1) nonenzymatic glycosylation (like Hb. A 1 c) : normally upto 8% of albumin (강동경희대병원 11 -16%) : hyperglycemia upto 25% 2) 짧은 반감기(17일) -> 수 주 동안의 혈당조절 평가에 이용 (Hb. A 1 c : 수개월) 3) Useful in hemolytic anemia (shortened RBC survival -> unreliable Hb. A 1 c) 4) Unreliable : protein losing nephropathy

Albumin - Measurement : with total protein (A/G ratio) Elevation Depression Dehydration Dilution (IV fluids) Prolonged application of tourniquet Loss of albumin (ascites, protein losing nephropathy or enteropathy) Impaired synthesis (hepatic dis. , malnutrition, malabsorption) cf) Prolonged application of tourniquet : venous backup force -> water & small solutes : out of intravascular space -> concentrating cellular elements, micellar forms of LP, proteins (albumin. . ) - Ca & Mg ions : bound to albumin, 농도 저하시 Ca & Mg 농도 저하 - Negative acute phase reactant - Compensation for decreased albumin (intravascular oncotic pressure 유지) Cirrhosis - polyclonal increase of Igs in γ-fraction (β-γ bridging) Nephrotic syndrome – increase of α 2 -MG - Albuminuria : 신장학

α 1 -Antitrypsin (AAT) - Major component of α 1 globulin fraction - Protease inhibitor : inactivation of trypsin (other serine protease inhibitors) (protection of tissue from enzymes (elastase) of inflammatory cells) - acute phase reactant, estrogen - 단백소실질환에서 감소 - AAT deficiency : hereditary disorder - lung(emphysema) , liver(cirrhosis) α 2 -Macroglobulin (AMG) - α 2 globulin fraction, protease inhibitor - 혈장에 있는 가장 큰 비면역글로불린 단백 (MW 725 k. Da) - Nephrotic syn. : 10배 이상 증가 (upto 2 -3 g/d. L), oncotic pressure (other LMW protein : loss into urine)

Haptoglobin - α 2 globulin fraction - 적혈구에서 유리되는 Hb과 결합하여 체내 iron과 단백 저장 유지 - half-life : 약 4일 but, Hb-haptoglobin complex - 수분 내에 제거(RES) - Decreased in hemolysis : 용혈성 수혈부작용, 화상, 자가면역성 용혈성빈혈… in impaired hepatic synthetic capacity - APR : Rise in response to stress, infection, acute inflammation & tissue necrosis - 적색뇨 DDx : hemoglobinuria vs myoglobinuria (신장학) urine strip reagent occult blood test (+) without RBC in urine DDx points : serum haptoglobin, CK, LD isoenzyme

β-Lipoprotein (β-LP) - Low density lipoprotein (LDL) - VLDL & HDL : small intensity -> 다른 단백과 중첩되어 EP 상에서 식별되지 않음 - hypercholesterolemia : β-LP band intensity 증가 Transferrin - Major β-globulin, MW 79. 55 k. Da - 세포 내 혹은 mucosal ferritin으로 저장되어 있는 철을 골수로 운반 - Normal serum 200 -400 mg/d. L (measured by iron-binding capacity, IBC) - Pseudoparaproteinemia in severe iron def. Fibrinogen - γ (β 인접), 340 k. Da, 100 -400 mg/d. L in plasma, acute phase reactant - absent from normal serum (plasma EP vs serum EP) plasma EP : distinct fibrinogen band between β & γ regions - heparinized patient : 불완전한 clotting, fibrinogen band 관찰 가능 DDx with monoclonal band - 검체관찰 및 재검

Minor Components 1. Ceruloplasmin α 2 migrating globulin, 132 k. Da, Liver, Copper-binding protein : contains most of copper in plasma Important in iron metabolism 2. Gc-Globulin α 1 migrating globulin, 51 k. Da Group specific component globulin (vitamin D binding protein, DBP) Decreased in severe liver disease 3. Hemopexin 4. α 1 -acid protein 5. CRP (C-reactive protein)

CRP - Patient serum recovered from pneumococcal infection + C-polysaccharide of pneumococci visible flocculation - Other disorders… : increase in tissue necrosis highly sensitive acute phase reactant - MI, stress, trauma, infection, inflammation, surgery, tumor. . presumptive Dx of bacterial (very high) vs viral (low) monitoring of rheumatologic disease - MW 118 – 144 k. Da, γ-migrating (severe inflammatory response – DDx with monoclonal gammopathy) - cut-off ≤ 0. 5 mg/d. L (5 mg/L) - hs. CRP (high sensitivity CRP) 혈청 지질과 함께 cardiovascular events (stroke, MI) 위험도 예측 inflammation in atherogenesis cut-off (American Heart Ass. & US CDC&P) Low risk < 1. 0 mg/L Average risk 1. 0 -3. 0 mg/L High risk > 3. 0 mg/L