人工血液第 14 巻 第 3 号 2007 年 2 月

目

次
会告

……………………………………………………………………

70

総説

周術期／救急領域の赤血球輸血と人工酸素運搬体の展望-Hemoglobin Vesiclesの可能性- ……………………寺嶋克幸

71

Newer Concepts of Oxygen Transport and Regulation:
Relation to Vascular Physiology ……………Paul C. Johnson

79

Polyethylene Glycol Conjugated Albumin:
A New Generation Plasma Expander …Nanae Hangai-Hoger

84

Hemoglobin Vesicle Aids Recovery of Cardiac Function during
Ischemia-Reperfusion in Langendorff Perfused Rat Hearts…………………………………………………Motoaki Bessho 92

ARTIFICIAL BLOOD
Vol. 14 No. 3 February, 2007

Contents
Announcement ………………………………………………………………

70

Review：Transfusion Overview in Perioperative/Emergent Field andProspect of Artificial Oxygen Carriers-A Potential of Hemoglobin Vesicles- ……Katsuyuki Terajima

71

Newer Concepts of Oxygen Transport and Regulation:
Relation to Vascular Physiology ……………Paul C. Johnson

79

Polyethylene Glycol Conjugated Albumin:
A New Generation Plasma Expander …Nanae Hangai-Hoger

84

Hemoglobin Vesicle Aids Recovery of Cardiac Function during
Ischemia-Reperfusion in Langendorff Perfused Rat Hearts…………………………………………………Motoaki Bessho 92

会 告
第14回日本血液代替物学会年次大会
会 期：平成19年 6 月14日
（木）
、15日
（金）
会 場：慶應義塾大学三田キャンパス北館（予定）
大会長：半田 誠（慶應義塾大学 輸血・細胞療法部）
テーマ：人工血液の未来を語ろう○プログラム
・一般演題
・大会長シンポジウム：人工血液の将来展望
基調講演
人工酸素運搬体
人工（代用）血漿
人工血小板

・シンポジウム、パネル、ワークショップ（いずれも一部公募）
人工酸素運搬体の諸問題
新規リコンビナント血漿蛋白製剤（抗体、凝固因子を含む）の開発状況

・教育講演
血小板とリコンビナント活性化凝固Ⅶ因子（仮題、ノボ・ノルデイック・フ
ァーマ、新井盛夫）
赤血球の話（仮題、女子医大生化学、高桑雄一）

・特別講演：人工血液に期待する
医療者の立場から患者の立場から
行政の立場から

大会事務局：〒160-8582 東京都新宿区信濃町35
慶應義塾大学 輸血・細胞療法部
電話（代表）：03-3353-1211 （内線62123）（ダイヤルイン）：03-5363-3715
ファックス：03-3353-9706
メール：mhanda@sc.itc.keio.ac.jp

70

人工血液 Vol. 14 , No.3, 2006

総説周術期／救急領域の赤血球輸血と人工酸素運搬体の展望-Hemoglobin Vesiclesの可能性Transfusion Overview in Perioperative/Emergent Field
and Prospect of Artificial Oxygen Carriers-A Potential of Hemoglobin Vesicles寺嶋克幸，杖下隆哉，坂本篤裕
Katsuyuki Terajima, Takaya Tsueshita, Atsuhiro Sakamoto

和文抄録
医療の飛躍的な発展にも関わらず，周術期や救急領域では多くの赤血球輸血の機会がある．近年の輸血療法に関する多くの報告を基に，日本赤十字社が輸血療法の実施に関する指針を発表し，改訂を重ねてきた．輸血を行うには，急性貧血時の代償機構と輸血の副作用を知らねばならない．輸血に対する安全性への努力はなされてきたが，根本的な問題の解決には至っていない．そこで，いくつもの人工酸素運搬体が開発された．しかし，安全性や効果の面で，一部のヘモグロビ
ンを利用した人工酸素運搬体のみが本邦において前臨床応用段階で有望視されている．幸いにも，我々は最も臨床応用可能と思われるHemoglobin Vesiclesの安全性及び効果を研究する機会を得た．本稿では，麻酔や外傷治療における貧血への耐性と輸液・輸血後の予後，およびヘモグロビン利用酸素運搬体の可能性について，我々の調査結果を含め再考した．
Abstract
Despite the remarkable medical advances were made over recent years, erythrocyte transfusions are often still required
during emergency care and the perioperative period. Several recent reports on blood transfusion have prompted the Japanese
Red Cross Society to publish revised guidelines for implementing transfusion therapy. In order to perform this procedures, onemust understand both the compensatory mechanism that occurs during acute anemia and the potential adverse reactions totransfusion. Although great efforts have been made to ensure the safety of blood transfusions, some fundamental issues have
yet to be resolved. Consequently, several artificial oxygen carriers have been developed; however, in terms of their safety andeffectiveness, only those that utilize hemoglobin have shown potential at the pre-clinical stage in Japan. We are privileged tohave had the opportunity to study the safety and effectiveness of hemoglobin vesicles, which appear to have the greatest
potential for clinical application. The present paper reviews tolerance to anemia under anesthesia and during trauma treatment,
post-transfusion prognosis, and the potential application of hemoglobin-based oxygen carriers, with reference to the results of
our own survey of these topics.
Keywords
Hemoglobin-based oxygen carrier, multiple organ failure, hemorrhagic shock, transfusion, red blood cell, fluid resuscitation

はじめに
麻酔中や救命救急領域では，出血や急性貧血に対する輸血の機会が非常に多い．昨年９月，「輸血療法の実施に関する指針」
及び「血液製剤の使用指針」の改訂が行われた1）．輸血方法を見直す報告が近年増加し，従来の輸血方法に対する批判も含まれている．特に，集中治療領域における輸血方法は患者予後に強く影響を及ぼす可能性がある．一方，血液代替物が開発されて75年が経過した．最近10年の進歩はめざましく，人工酸素運

日本医科大学 麻酔科学教室 〒113-8603 東京都文京区千駄木1-1-5 Department of Anesthesiology, Nippon Medical School

1-1-5, Sendagi,

Bunkyo-ku, Tokyo, 113-8603, JAPAN
論文受付 2006年６月16日 論文受理 2006年７月19日

ARTIFICIAL BLOOD Vol. 14 , No.3, 2006

71

搬体は一部の国で臨床応用や臨床治験されるまでになった．現
在，我が国でも数種類の人工酸素運搬体が動物試験されている．
この論文では，酸素運搬体としての赤血球輸血とヘモグロビン
利用の人工酸素運搬体に焦点を当て，麻酔／救急領域における赤血球輸血療法の適応と問題点，人工酸素運搬体利用による利点について我々の報告を含め概説する．

手術中の急性貧血に対する耐性
最も重要な赤血球の機能は，呼吸器（肺）から末梢組織への酸素運搬である．手術中のような院内出血は，晶質液や膠質液による循環血液量の補正後に必要な場合に対してのみ輸血が行われる．この時の輸血実施に対する判断は，生体の代償機能を超えるか否かの判断に基づく．この代償機能は，主に血行動態の変化とヘモグロビン酸素解離曲線の変化による 2）．循環血液量の保たれている急性貧血は，一回拍出量と心拍数の増加により心拍出量は増加する3）．血液粘稠度の減少は，静脈還流増加による前負荷の増加と後負荷を軽減する4）．そして，交感神経刺激は陽性変時・変力作用を来すことによる．従って，麻酔下の患者は，主に一回拍出量によって心拍出量を増加させる．全身的には，ヘモグロビン濃度が7 g/dLまでは酸素供給量は変化
しない3）．脳は酸素抽出率を増加することで急性貧血に対応するのに対し，心臓は酸素抽出率よりも酸素供給量の増加により代償する．微小循環に対しては血管床と血流の増大により酸素放出増加を可能にする2）．また，貧血による2,3-DPGの増加は，酸素解離曲線を右方変位させ，末梢での酸素放出を容易にさせる．
麻酔中の冠動脈疾患患者での循環血液量の保たれている中等度急性貧血は許容されるようである．冠動脈バイパス術を予定されている患者のヘモグロビン濃度13.9 ± 1.3 g/dLから9.3 ±1.0 g/dLへの血液希釈では，心筋虚血を示唆する心電図変化や局所壁運動異常，血行動態の変化は見られなかった5）．このような臨床研究での対象患者は，予後を改善するために周術期もβ遮断薬による治療を継続している6）．β遮断薬の使用の有無
は，急性血液希釈による心拍出量の増加を阻害しない7）．また，麻酔下の状況では陽性変時作用が見られないことも手術中のイベントの発生率を下げるかもしれない．さらに，オンポンプ冠動脈再建術患者のヘマトクリット値28％への急性血液希釈は，術後の血中トロポニンIやCK-MBレベルをコントロール群に比べ抑制し，カテコラミン必要量を低下させ術後不整脈発生率を低下した8）．急性冠動脈症候群患者では，30日死亡の予測因子としてヘマトクリット値25％以上での輸血開始が挙げられた9）．
ただし，この研究は３つの急性冠動脈症候群に対する大規模研
究データに基づくレトロスペクティブな研究であることを考慮しなければならない．
血液希釈によるヘモグロビン濃度6 g/dL以下の低下は，認知機能を低下させる10-12）．ヘモグロビン濃度5 g/dLまでの低下による認知機能の低下は可逆的であり，新鮮血や保存血の違いに関わらずヘモグロビン濃度7 g/dLまで輸血することで回復する．また，吸入酸素濃度の増加によっても回復する．その障害時には，大脳誘発電位の一種である事象関連電位に見られる，

72

刺激からの陽性波の発生までの時間，すなわちP300潜時の延長として表される可能性がある．
軽度から中等度の血液希釈は肺の換気血流不均等を改善させ 13），
肺高血圧を伴う慢性閉塞性肺疾患患者に対して肺動脈圧を減少させる14）ことが示唆されている．血液粘度の低下や心拍出量の増加に加え，NOの増加による肺血管抵抗の減少が生じるためと考えられる．これにより，動脈血酸素分圧/吸入酸素濃度比は増加する．
腎血流量と血液分布は中等度血液希釈に影響を受けず，尿量と尿中ナトリウム排泄率は増加する可能性がある 15）．同様に，
ヘマトクリット20 ± 1％までの血液希釈は肝臓や小腸，膵臓の血流を増加させる 16）．肝臓は，肝動脈の血流が86％増加し，門脈血流は28％増加した．小腸粘膜血流も増加した．ICG血管
内半減期は短縮し，GPTは減少した．すなわち，肝機能を障害しないようである．
血液希釈による酸素供給量の重篤な低下は，主に乳酸値，重炭酸イオン，過剰塩基（BE），酸素抽出率，呼気二酸化炭素濃度，脈圧，心係数，収縮期血圧に影響を及ぼす17）．臨床的に輸血を行う根拠の一つとして利用出来る可能性がある．

プレホスピタル出血性ショック
我々の関わるもう一つの分野に，救命救急や集中治療がある．
救命救急の患者の多くは院外発症である．すなわち，院外発症の急性出血性ショック患者は通報により駆けつけた救急救命士
により初期治療が施され，直ちに近くの救急対応病院に搬送される．このプレホスピタルの出血性ショックの特徴は，全ての血液成分が循環血液量とともに同程度に失われることである．
残念なことに，我が国での救急救命士によるfluid resuscitation（輸液蘇生）は行われていない．欧米諸国では，患者搬送時間の問題もあるが，この救急救命士による初期輸液蘇生は急性出血性ショック患者の救命の一役をになっている．初期輸液蘇生の方法は，未だ発展途中である18）．必要な止血処置とともに行
われるプレホスピタルでの古典的輸液蘇生法は，大量の晶質液投与による循環維持をすることである．その後のインホスピタ
ルで必要があれば，輸血が行われる．現在臨床治験中や開発中の人工酸素運搬体の役割の一つには，このプレホスピタルへの治療限界への挑戦が挙げられる．

人工酸素運搬体の過去の治験から学ぶ
修飾Hb４量体のヘムアシストは，第３相臨床試験で予期せず失敗に終わった19）．このヘモグロビン利用酸素運搬体は，臨床治験前に少量投与で循環動態に強く影響することが分かっていた20，21）．生来のHbと同じ様に，少量投与で，肺血管抵抗や体血管抵抗を増加し心拍出量を低下させた．これは，NOのスカ
ベンジ22-24）とエンドセリン放出増加，アドレナリン受容体の感
受性増大25-27），二次的な動脈壁ずり応力の減少によるものと考えられている．出血性ショック患者へのプレホスピタルでの初期蘇生として，強力な血管収縮薬の投与は死亡率を増加させる
結果となった．

人工血液 Vol. 14 , No.3, 2006

Hemoglobin-Vesicles（HbV）の循環動態と臓器酸素化へ及ぼす影響
HbVは我が国で開発されたHBOCである．現在，開発した早稲田大学理工学部や慶応大学医学部を中心に精力的に研究活動を行っている．昨年，我々の教室でも，動物モデルでのHbVを用いた輸液蘇生での全身循環及び臓器酸素化への影響を調査した28）．
対象動物は，生後12 - 14週齢の雄New Zealand white rabbitとした．早稲田大学理工学部の協力を得て，ウサギ赤血球とHbV（委託製造先，㈱オキシジェニクス）の酸素解離曲線を得た（Fig. 1.）．麻酔下に小開頭と開腹，大腿部切開し，脳／肝
臓／腎臓／骨格筋に酸素電極を挿入した．ベースライン測定後，平均動脈圧が30-35 mmHgとなる様に脱血を行った．その後，脱血量と等量の５％リコンビナントアルブミン溶液＋HbV溶液（HbV＋alb群），５％リコンビナントアルブミン溶液（ニプロ㈱提供）（alb群），乳酸リンゲル液（LR群），さらに脱血の３倍量の乳酸リンゲル液（3XLR群）で急速輸液蘇生を行った．測定
項目は，脱血量，動脈圧，中心静脈圧，心拍出量，組織酸素分圧とした．脱血量は群間に差がなかった．血行動態の変化をTable 1.に示す．LR群は蘇生終了15分後より，3XLR群は1時間
後よりベースラインに比べ血圧が低下した．心拍出量はLR群と3XLR群ともに２時間後に低下した．それに対し，HbV＋alb
群とalb群の心拍出量は蘇生後に回復し，実験期間中維持された．蘇生後15分でのHbV＋alb群の計算された全身血管抵抗は，他の群と差がなかった．HbV投与による蘇生後の組織酸素分圧
は，他の蘇生群と比べ低下しなかった．脳や腎臓では，蘇生後15分以降ではLR群に比べ高く維持された．BEや血中乳酸値も他群に比べより早く回復した．これらの結果は，急性出血性シ
ョックに対するHbV溶液の投与が，生体内で血管収縮による全身や局所の血管抵抗の増大を来さず，心拍出量と組織酸素分圧を回復させることを示唆する．さらに，全身の循環と酸素化改善により乳酸値とBEを早期に回復させることが示唆された．

Fig. 1. The oxygen equilibrium curves for a）rabbit Hb and b）HbV.
Table 1. Hemodynamic variables in New Zealand white rabbits after inducing hemorrhagic shock（HS）by withdrawing blood andstabilization for 30 min. Animals were resuscitated using the same volume of HbV/rHSA, rHSA or RL, or using 3ｴRL, ove
15 min（RES）. The hemodynamic variables were measured again after 15 min, 1 h and 2 h. *Significant difference frombaseline（p<0.05）. †Significant difference from the RL group（p<0.05）. All values are presented as the mean ± SD（n = 6）.

BaselineHS
Mean arterial pressure（mmHg）
33 ± 1*85 ± 10
HbV/rHSA
32 ± 3*89 ± 11rHSA
34 ± 1*78 ± 10RL
31 ± 3*80 ± 17
3XRL
Central venous pressure（mmHg）
3.0 ± 0.9*
4.5 ± 1.2
HbV/rHSA
3.2 ± 1.2*
5.2 ± 1.7rHSA
2.7 ± 0.8*
6.2 ± 1.3RL
2.8 ± 1.0*
4.3 ± 1.0
3XRL
Cardiac Index（l･minute-1･m-2）
2.9 ± 0.7
HbV/rHSA
2.7 ± 0.8
rHSA
2.6 ± 0.9RL
2.9 ± 0.5
3XRL
Systemic vascular resistance（dyne･second･cm-5）
HbV/rHSA 1579 ± 738
1834 ± 494
rHSA1559 ± 564
RL1575 ± 465
3XRL

ARTIFICIAL BLOOD Vol. 14 , No.3, 2006

30 minRES

15 min1h2h

43 ± 3*36 ± 2*41 ± 9*38 ± 7*88 ± 10†68 ± 1469 ± 967 ± 1290 ± 16†76 ± 9†56 ± 13*62 ± 1790 ± 17†77 ± 9†51 ± 10*48 ± 20*90 ± 7†80 ± 9†54 ± 20*46 ± 16*2.8 ± 1.5*
3.0 ± 1.3*
2.8 ± 0.4*
2.8 ± 1.2*

5.8 ± 1.5
5.0 ± 1.7
4.7 ± 1.4
5.5 ± 0.8

5.8 ± 1.5
4.8 ± 1.5
4.0 ± 1.7*
4.8 ± 1.2

4.2 ± 1.2
4.7 ± 0.5
3.8 ± 0.8*
3.7 ± 0.5

4.7 ± 1.2
4.2 ± 0.8
3.5 ± 1.0*
2.8 ± 1.0*

1.2 ± 0.4*
1.0 ± 0.2*
1.1 ± 0.4*
1.1 ± 0.2*

2.9 ± 0.2†
2.7 ± 0.7
1.8 ± 0.4
2.6 ± 0.9

3.2 ± 0.8†
2.3 ± 0.5†
1.3 ± 0.3
1.9 ± 0.6

2.8 ± 0.4†
2.4 ± 0.6†
1.1 ± 0.3*
1.4 ± 0.4*

1839 ± 420
1956 ± 3942012 ± 695
1868 ± 370

1516 ± 294
1600 ± 569
1578 ± 509
1374 ± 334

1498 ± 440
1820 ± 549
1993 ± 587
1420 ± 717

1516 ± 294
1600 ± 569
1578 ± 509
1374 ± 334

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Fluid resuscitationの歴史29）
プレホスピタルでの輸液蘇生の進歩は，残念なことに大きな戦争ごとに見られるようである．戦場では医療に対して障害がある．初期の蘇生処置から根治的治療までの時間，治療薬の保存や運送面において効果的な初期治療のための方法と薬剤が求められる．
第一次世界大戦の時は，まだ術前のfluid resuscitationが導入させておらず，多くの兵を失った．第二次世界大戦や朝鮮戦争の時は，膠質液の投与と保存血の投与による蘇生が始まった．
初期生存率の改善をもたらしたが，急性腎不全のために多くの死傷者が出た．ベトナム戦争の時には，以前の経験と大量等張晶質液による初期輸液蘇生が見直され，それまでのコロイド輸液から変わった．その結果，死亡率と急性腎不全の発生率は減
少したが，adult respiratory distress syndrome（ARDS）の発生率とそれによる死亡率が増加した．1970年代より当時の先端医療技術とモニタリングなどの特徴を持つ集中治療室が発達し，ARDSでの死亡率は減少した．しかし， 多臓器不全 という現在でも外傷患者の死亡原因として最も重大な問題が明白となった．
後に，初期蘇生輸液の晶質液の種類についての研究が報告された30）．中等度出血性ショックモデルに対する輸液／輸血蘇生
では，生理食塩水を用いた群は乳酸リンゲル液での蘇生群に比べ，アシドーシス（7.14 ± 0.06対7.39 ± 0.04）と死亡率の増加（50％対0％）が観察された．

Small volume fluid resuscitationと permissive hypotension
急性ショック患者の主な目的の一つには，十分な酸素供給量に回復することである．酸素供給量に関わる要素は，ヘモグロ
ビン濃度，酸素飽和度，心拍出量を含む．そのため，心拍出量を改善するための充分な輸液量と酸素運搬体の補充が必要となる．院内蘇生の最初の24時間の酸素供給量インデックスの目標
は，現在のところ500 mL/minute/m2と報告された31）．それ以上に酸素供給量を増加させても酸素消費量を増加させず，血中乳酸値は増加したままとなることがある．これは，末梢のミトコンドリア機能障害を示し，多臓器不全に導く32）．また，過剰な輸液や輸血は，血圧上昇や凝固能への影響による出血量の増加や，低体温，アシドーシス，脳浮腫やARDS発生率の増加のような組織浮腫の可能性がある33）．鈍的外傷による出血源のコ
ントロールされていない患者に対する等容量輸液蘇生は，このような危険を含んでいる．
このような患者の輸液蘇生に対し，輸液量を制限する戦略や低血圧を維持させる戦略が始まった34）．少量高張食塩水は大量
晶質液と同等の心拍出量と血漿増量効果，血管内皮の膨化を防ぐことでの末梢循環改善効果，生存率改善が示された35）．これは，心原性ショックに対しても末梢循環改善効果をもたらす36）．
しかし，この循環改善効果は出血量の増加をもたらした．高張
食塩水の研究のメタアナリシスでの結果は，標準的な晶質液での輸液蘇生後の生存率を改善しなかった37）．この生存率改善効
果は，デキストランを添加することで可能性があることが示唆

74

された 37，38）．さらに，多施設試験での蘇生後の臓器保護効果39）
やpermissive hypotensionによる蘇生後の予後を改善する効果40）が示された．特に，ショックを伴う閉塞性頭部外傷患者では脳浮腫を軽減し頭蓋内圧を減少し，脳還流圧を上昇することが示された41）．さらに，この高張食塩水の蘇生は，出血性ショックや虚血再還流において炎症反応を減少させることが示唆された42-44）．

HbVの凝固機能への影響
出血性ショックに対する輸液蘇生時には，大量出血による凝固因子の流失，凝固機能亢進による消費，輸液蘇生に用いられる溶液の影響による出血量の増大が生じる可能性がある．さらに，前述の様に，末梢循環の改善効果のある溶液は，出血量を増加させる．出血性ショックモデルに対するHbV溶液を用いた
輸液蘇生は，全身末梢血管抵抗に影響を及ぼさないようである28）．そこで我々は，HbV溶液による血液希釈が凝固機能に与える影響をin vitroで調査し，報告した45）．
健康成人男性のボランティア７人を対象とした．採血された
血液サンプルを，直ちに乳酸リンゲル液，または，生理食塩水に分散した10％HbV溶液で，それぞれ，乳酸リンゲル液，または，生理食塩水が血液サンプルに対して０％，20％，40％，
60％，80％になる様に希釈した．乳酸リンゲル液で希釈した血液は，フィブリノーゲン濃度，プロトロンビン時間，活性化部
分トロンボプラスチン時間を測定した．また，全ての血液サン
プルまたは希釈された血液はSONOCLOT（tm）（Sienco
Company, Morrison, CO） で 凝 固 機 能 を 測 定 し た ．
SONOCLOT（tm）は，血小板機能や血漿成分凝固因子，細胞成分凝固機能を，37℃における血液の粘度抵抗の経時的な変化に基づいてクロット形成と線溶現象を測定することで評価するものである46，47）．希釈の程度に比例して，フィブリノーゲン濃度は低下した（Fig. 2a.）．プロトロンビン時間と活性化部分ト
ロンボプラスチン時間は，希釈の程度以上に延長する傾向を認めた（Fig. 2b, 2c.）．SONOCLOT（tm）による測定値を示す（Fig. 3.）．60％希釈まではHbV溶液は乳酸リンゲル液に比べて差はなかった．しかし，80％希釈ではHbV溶液での希釈はSonACTを延長させた．HbV溶液による高度の血液希釈は，乳酸
リンゲル液に比べ抗凝固作用を示す可能性が示唆された．ただし，60％希釈までの場合，ヒドロオキシエチルスターチに比べ影響が少ない可能性がある46，47）．我々の研究では，60％までの希釈では，乳酸リンゲル液とHbV溶液のSONOCLOT（tm）による測定値は差がなかった．すなわち，HbV溶液で高度希釈を行うような特殊な場合を除き，臨床応用される状況下では凝固異常が問題となる可能性は低いことが示唆された．

赤血球輸血による外傷後多臓器不全と肺障害
輸血を必要とする外傷患者の蘇生後のMOFの発生率を低下させることは，現在の出血性ショック治療の主な関心の一つである48）．Injury Severity Score（ISS）15以上の513人の外傷患者でのコホート研究では，外傷後12時間以内の6単位以上の輸血はMOF発生の独立危険因子であった49）．また，最初の外傷後

人工血液 Vol. 14 , No.3, 2006

2a

3a

2b

3b

2c

3c

Fig. 2. Fibrinogen concentrations, prothrombin time and the activatedpartial thromboplastin time of diluted samples with isotonic
Ringer's lactated solution（RL）or 10 g/dL Hemoglobin-Vesicles
solution（HbV）. Fibrinogen concentrations were decreased ininverse proportion to the dilution rate（2a）. The prothrombin time
and the activated partial thromboplastin time were in proportion
to the square or cube of the dilution rate（2b, 2c）.

６時間以内に投与された赤血球の保存期間の長さ（古さ）が外傷後MOFの発生に対する独立危険因子であった 50）．外傷後
MOF発生のハイリスク患者では，循環中の好中球はプライミングされていてアポトーシスに抵抗性を示し51），血管内皮細胞のIntercellular adhesion molecule-1（ICAM-1）の発現が増加する．この原因の一つには，貯蔵赤血球バッグに混入する白血球があげられる52）．そして，保存血の保存期間が長くなればなるほど，白血球や血小板由来の活性化物質は増加することが報告された53）．さらに，貯蔵赤血球中の血漿と脂質は実験的に急ARTIFICIAL BLOOD Vol. 14 , No.3, 2006

Fig. 3. SONOCLOT values of diluted samples with isotonic Ringer's
lactated solution（RL）or 10 g/dL Hemoglobin-Vesicles solution（HbV）. Activated coagulation time（ACT）and clot rate（CR）was
prolonged in the 40％ and 60％ dilution with LR and HbV
compared with baseline values, but there was no significancebetween the two groups（3a, 3b）. In the 80％ dilution by both
solutions, ACT and time to peak was affected in comparison withbaseline（3a, 3c）.

性肺障害を生じることが示された54）．

HbVの肺への影響
我々は，出血性ショックモデルに対するHbV投与後の肺への影響を調査し報告した55）．週齢10-13週の雄Sprague-Dawleyラット72匹を対象とした．１）（単純投与）まず，36匹を自発呼75

吸でセボフルレン麻酔下に，11.2 mL/kgの生理食塩水，または，生理食塩水に分散した10％HbV溶液を30分で経静脈投与した．投与後２時間，24時間，72時間に犠牲死させ，肺を摘出した．２）（出血性ショック後輸液蘇生）次に，36匹を自発呼吸でセボフルレン麻酔下に，28 mL/kgの血液を20分以上かけて脱血し，安定させた．その後，脱血量と等量の生理食塩水に分散した10％HbV溶液（HbV群），または，脱血量の３倍量の乳酸リンゲル液（RL群）で輸液蘇生を行った．ベースラインと脱血直後，蘇生後１時間，２時間後の血行動態とヘマトクリッ
ト値，BE，血中乳酸値を測定した．輸液蘇生後2時間，24時間，72時間に犠牲死させ，肺を摘出した．肺のhypoxic inducible
factor 1 alpha subunit（HIF-1α），tumor necrosis factor alpha（TNF-α），interleukin-6（IL-6），heme oxygenase- 1（HO-1），
inducible nitric oxide synthase（iNOS），intercellularadhesion molecule 1（ICAM-1）のmRNAの発現をRT-PCR法で測定した．脱血直後の平均動脈圧は，両群間で差はなかった（RL群33 ± 4 mmHg，HbV群30±3 mmHg）．蘇生後のヘマト
クリット値は，1時間後，2時間後共に，HbV群の方が有意に高かった．蘇生後1時間後の血中乳酸値はRL群の方が有意に高かった．単純投与後は，いずれの測定時点でも，我々の測定したmRNAの発現には影響しなかった．HbV群は，RL群に比べ，投与２時間後のHIF-1αとIL-6が抑制されており，HO-1とTNFαがより発現していた．また，72時間後のHO-1の発現が増加しており，IL-6の発現が抑制されていた．ICAM-1は単純投与
でも出血性ショック後輸液蘇生でもHbV投与に影響を受けず，肺への血管内皮細胞への影響はないことが示唆された．また，Hb自体の影響として輸液蘇生直後のHIF-1αの発現抑制とHO-1発現増加が見られたと思われる．

海外でのHBOCsの研究報告と今後の展望
現在，南アフリカでは重合HbであるHemopure（Biopure
Corp., Cambridge, MA）が急性出血に対して臨床利用されている．PolyHeme（Northfield Laboratory, Evanston, IL）は，
大規模な第３相臨床研究が行われている56）．基礎研究でも，外傷後MOFモデルとして２イベントモデルを作成し精力的な研究が行われている57）．最終的な目的は，従来の赤血球輸血に比べてMOFやARDSの発生率を低下させ生存率を改善することだろう．しかし，臨床研究では対象患者の均一化や遺伝子多型
性による反応の違い，フィジシャンの違い，目的の難しさなど様々な問題があり長い時間を費やす．HbVを含む人工酸素運搬体の臨床応用は，重大な副作用を来さず，緊急時の酸素運搬体としての簡便な適応に加えて，外傷直後の赤血球輸血を減らす
ことによるその後の急性肺障害や多臓器不全への発展を予防する可能性がある．血液成分を考えれば，酸素運搬体だけの投与は研究のプロトコルに制限があり，臨床適応するのが難しいと考えられる．このような困難にも関わらず，利用価値のある製品が十分な改良と適応を熟考された上で，臨床応用可能となる
ことはきわめて合理的であると考えられる．我が国で開発されたHbVや海外で臨床治験中や臨床応用されているHBOCsの緊

76

急時や手術中の患者に対する影響を理解することは，新たな種類の特徴ある人工酸素運搬体の有効な臨床応用を可能にするだ
ろう．

謝辞
本研究は，厚労省科学研究費補助金医薬品・医療機器等レギュラトリーサイエンス総合研究事業「救急・災害医療に利用可
能な人工赤血球の開発に関する研究（研究課題番号：H17-医薬-072）の分担研究として実施した．研究を進めるにあたり
HbVの供与ならびに研究の機会を与えて頂いた研究班主任研究者・慶應義塾大学医学部外科教授四津良平先生，ウサギ赤血球
ならびにHbVの酸素解離曲線を提供頂きました早稲田大学理工学総合研究センター顧問研究員土田英俊先生ならびに助教授酒井宏水先生に深く感謝申し上げます．

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Efficacy of hypertonic 7.5％ saline and 6％ dextrane-70 intreating trauma: A meta-analysis of controlled clinicalstudies. Surgery 1997;122:609-616.
38. Smith GJ, Kramer GC, Perron P. A comparison of several
hypertonic solutions for resuscitation of bled sheep. J Surg
Res 1985;39:517-528.
39. Mattox KL, Maningas PA, Moore EE, Mateer JR, MarxJA,
Aprahamian C, Burch JM, Pepe PE. Prehospital
hypertonic saline/dextran infusion for post-traumatic
hypotension. The U.S.A. Multicenter Trial. Ann Surg
1991;213:482-491.
40. Burris D, Rhee P, Kaufmann C, Pikoulis E, Austin B, Eror
A, DeBraux S, Guzzi L, Leppaniemi A. Controlled
resuscitation for uncontrolled hemorrhagic shock. J
Trauma 1999;46:216-223.
41. Shackford SR. Effects of small-volume resuscitation on
intracranial pressure and related cerebral variables J
Trauma 1997;42:S48-S53.
42. Coimbra R, Hoyt DB, Junger WG, Angle N, Wolf P, Evers
MF, Badellino MM, Shackford SR, Simon RJ, Barie PS.
Hypertonic saline resuscitation decreases susceptibility tosepsis after hemorrhagic shock. J Trauma 1997;42:602-607.
43. Zallen G, Moore EE, Tamura DY, Johnson JL, Biffl WL,
Silliman CC. Hypertonic saline resuscitation abrogatesneutrophil priming by mesenteric lymph. J Trauma
2000;48:45-48.
44. Rotstein OD. Novel strategies for immunomodulation after
trauma: revisiting hypertonic saline as a resuscitation
strategy for hemorrhagic shock. J Trauma 2000;49:580-583.
45. Tsueshita T, Terajima K, Takeda S, Sakamoto A, Ogawa
R. In vitro effect of hemoglobin-vesicles solution on
coagulation using sonoclot analysis. International

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Anesthesia Research Society 79th Clinical & Scientific
Congress. Honolulu, U.S.A.
46. Konrad C, Markl T, Schuepfer G, Gerber H, Tschopp M.
The effects of in vitro hemodilution with gelatin,hydroxyethyl starch, and lactated Ringer'ssolution onmarkers of coagulation: an analysis using SONOCLOT.
Anesth Analg 1999;88:483-488.
47. Konrad CJ, Markl TJ, Schuepfer GK, Schmeck J, Gerber
HR. In vitro effects of different medium molecularhydroxyethyl starch solutions and lactated Ringer's
solution on coagulation using SONOCLOT. Anesth Analg
2000;90:274-279.
48. Moore EE, Johnson JL, Cheng AM, Masuno T, Banerjee
A. Insight from studies of blood substitutes in trauma.
Shock 2005;24:197-205.
49. Moore FA, Moore EE, Sauaia A. Blood transfusion. An
independent risk factor for postinjury multiple organ
failure. Arch Surg 1997;132:620-625.
50. Zallen G, Offiner PJ, Moore EE, Blackwell J, Ciesla DJ,
Gabriel J, Denny C, Silliman CC. Age of transfused bloodis an independent risk factor for postinjury multiple organ
failure. Am J Surg 1999;178:570-572.
51. Biffl WL, Moore EE, Zallen G, Johnson JL, Gabriel J, Offner
PJ, Silliman CC. Neutrophils are primed for cytotoxicity
and resist apoptosis in injured patients at risk for multiple
organ failure. Surgery 1999;126:198-202.
52. Bordin JO, Heddle NM, Blajchman MA. Biologic effects ofleukocytes present in transfused cellular blood products.
Blood 1994;84:1703-1721.
53. Nielsen HJ, Reimert CM, Pedersen AN, Brunner N,
Edvardsen L, Dybkjaer E, Kehlet H, Skov PS. Timedependent, spontaneous release of white cell- and plateletderived bioactive substances from stored human blood.
Transfusion 1996;36:960-965.
54. Silliman CC, Voelkel NF, Allard JD, Elzi DJ, Tuder RM,
Johnson JL, Ambruso DR. Plasma and lipids from stored
packed red blood cells cause acute lung injury in an
animal model. J Clin Invest 1998;101:1458-1467.
55. 杖下隆哉，寺嶋克幸，坂本篤裕ほか 人工赤血球の安全性に関する研究 第53回日本麻酔科学会総会 神戸
56. Moore EE, Cheng AM, Moore HB, Masuno T, Johnson JL.
Hemoglobin-based oxygen carriers in trauma care:scientific rationale for the US multicenter prehospital trial.
World J Surg 2006
57. Aiboshi J, Moore EE, Ciesla DJ, Silliman CC. Bloodtransfusion and the two-insult model of postinjury
multiple organ failure. Shock 2001;15:302-306.

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Review

Newer Concepts of Oxygen Transport and Regulation:
Relation to Vascular Physiology
Sangho Kim, Marcos Intaglietta and Paul C. Johnson (1)Introduction
Oxygen transport and blood flow regulation are linked by avariety of mechanisms but the linkage is complex and basedon the interactions of a number of seemingly unrelated
factors. The purpose of this presentation is to provide an
overview of this area. As will become apparent in thispresentation, the arteriole, which is the primary regulator ofblood flow, is acted upon by a variety of stimuli that
determine its degree of contraction and relaxation under
different conditions.

Chemosensitive Mediation of Blood Flow
The metabolic hypothesis
The simplest relationship between the rate of blood flow
and the rate of oxygen transport, is the metabolic
hypothesis 1）as shown in Figure 1. This is based on theconcept that arterioles possess chemosensitive properties.

Fig. 1. Schematic diagram of a model of metabolic regulation of blood
flow. Details are presented in the text. From（1）by permission.

According to this hypothesis the tissues are constantly
producing metabolites that cause vasodilation of thearterioles. If oxygen delivery is adequate, a vasodilator
product of aerobic metabolism, CO2 , will be produced as afunction of the rate of aerobic metabolism. In turn, as
carbonic acid is formed there will be an increase in H+, which
also has potent vasodilator effects/ In this case the rate ofblood flow will be determined by the concentration of CO2
acting on the arterioles. That concentration will be directly
dependent on the ratio of oxygen consumption to blood flow.
The attractive feature of this hypothesis is its simplicity andits ability to provide a direct linkage between the rate ofoxidative metabolism and blood flow. And in fact, certain
vascular beds such as myocardium2）and brain3）are sensitive
to CO2 and this hypothesis is a plausible possibility to explain,at least in part, blood flow regulation in those organs.
However, in other vascular beds such as skeletal muscle
there is a also direct relationship between blood flow andoxygen consumption but detailed studies have failed to reveal
a significant role for CO2 in flow regulation4）. These findings
have led researchers to examine other possible mediators
which might be produced when the oxygen levels are
sufficient overall but in localized areas are insufficient, leading
to a shift to anaerobic metabolism in the parenchymal cells.
According to this hypothesis it is proposed that undernormal conditions when oxygen levels are sufficient for mostregions of the tissue there are certain tissue areas in which
the oxygen supply is inadequate5）causing these areas to rely
on mediators such as adenosine and H + produced byanaerobic metabolism. Secondarily, depolarization of theparenchymal cells will cause release of K+ from these cells.（Asmall increase in extracellular K+ has been shown to increase
K + conductance of the plasma membrane, leading tohyperpolarization and relaxation of vascular smooth muscle.）
A logical location for a tissue area of anaerobic metabolism isthe venous end of the capillary network where the oxygen

Department of Bioegineering, University of California, San Diego, La Jolla, CA 92093-0412（1）To whom correspondence should be sent. Email address: pjohnson@bioeng.ucsd.edu
論文受付 2006年12月６日 論文受理 2006年12月12日

ARTIFICIAL BLOOD Vol. 14 , No.3, 200679

concentration in the blood flowing through the capillary
network reaches its lowest level. In addition, it is well known
that flow rate of blood through the microcirculatory vessels
and hematocrit in these vessels are both highly variable,
lending credence to the possibility that such areas might
normally be present. If they are present, the release ofvasodilator products of anaerobic metabolism would provide a
feedback system in which these vasodilator agents would
diffuse to nearby arterioles and adjust the state of contractionof the vascular smooth muscle according to the degree ofanaerobiosis present.
This hypothesis has been tested in our laboratory bymonitoring the level of NADH fluorescence in microregions of
the tissues 15 to 25 μm diameter in the vicinity of thecapillary network of skeletal muscle as blood flow is reduced
or stopped completely for a period of time. A rise in NADH
level is taken as an indication of a shift from aerobic toanaerobic metabolism. We found that with complete occlusionof blood flow there was no change in NADH level for an
average of 45 s and there was no difference in this latent
period between the arteriolar and venous ends of thecapillary network 6）. In these studies NADH increases at theearliest in 10 to 15 s after occlusion. When, instead ofcomplete stoppage, blood flow was reduced by 50%, aminimum of 30 s passed before a detectable increase in
NADH occurred 7）. Both of these findings suggest that undernormal conditions there are no microregions of the tissue inthe vicinity of the capillaries that are hypoxic or on the vergeof hypoxia.
While these findings do not support the concept that the
metabolic state of the parenchyma contributes to blood flow
regulation under resting conditions, there is evidence that it
is important when oxygen levels become inadequate. We
have found that the magnitude of reactive hyperemia（theperiod of increased flow following a period of blood flow
stoppage）is related to the duration of stoppage（unpublishedfindings）. Interestingly, as the period of stoppage is increased
from 5 s to 45 s, the amplitude of the reactive hyperemia
following restoration of flow increases up to 4 times thecontrol flow but the duration remains relatively constant at
about 70s. However, when the flow stoppage exceeds 45 s
NADH fluorescence of the tissue begins to increase and theduration of hyperemia increases as a function of the period of
stoppage. This finding provides clear evidence that a shift toanaerobic metabolism in skeletal muscle can produce
vasodilator metabolites, possibly including H +, lactate andadenosine, and increase the duration of reactive hyperemia.
Whatever the specific mediators may be, it appears that theyremain in the tissues until they are reincorporated into the80

energy supply or washed out by the blood stream. We havesome evidence in these studies that washout is more
important than reincorporation.
Oxygen-dependent mediators at the arteriolar level
To this point we have focused on factors that may produce
vasodilation in relation to the metabolic requirements of theparenchymal cells in the tissue. There are, however, avariety of oxygen-dependent mechanisms at the level of thearterioles that may play key roles in blood flow regulation.
These mechanisms adjust the contractile state of the smoothmuscle in accordance with the local PO2.
Microcirculatory studies have revealed that elevation of theoxygen level in suffusing fluid over a surgically exposed
tissue reduces blood flow due to constriction of thearterioles8）. Several mechanisms may be involved as recently
reviewed by us9）. It appears that this response may be due toin part to release of a vasoconstrictor cytochrome P450
metabolite of arachidonic acid, 20-HETE, from the arteriole
when arteriolar PO2 is elevated. There is also evidence that
release of vasodilator prostaglandins from the endothelium
increases when PO 2 is reduced. Additionally, it has beenshown that nitric oxide release from the endothelium
increases when oxygen falls below normal levels. Finally, it
appears that the endothelium also releases the vasodilator
adenosine during hypoxia.
An additional mechanism that may be important is basedon the finding that deoxygenation of the red cell may cause
relaxation of the arterioles due to release of ATP which acts
on purinergic receptors. It has been proposed also that as
the red cell is deoxygenated in passage through the capillary
bed ATP is released and acts on the venular endothelium torelease vasodilator prostaglandins which would in turn
diffuse to nearby arterioles10）. This would provide a feedback
arrangement in which greater oxygen extraction from theblood at the capillary level would automatically lead toarteriolar vasodilation and return of blood flow towardnormal levels. The overall effect of the oxygen-dependentchemical mediators elicited at various levels as describedabove is summarized in Figure 2. In this figure the roles of
the red cell, endothelium and the parenchymal cells andspecific mediators in flow regulation are shown.

Mechanosensitive mechanisms
In addition to the chemosensitive mechanisms describedabove there is a separate class of important,mechanosensitive mechanisms that respond to physicalstimuli. These are the myogenic response which is due tocircumferential tension in the vascular smooth muscle

人工血液 Vol. 14 , No.3, 2006

to its surroundings, may act as tension sensors to mediate the
myogenic response, in part by causing depolarization in the
fashion described above.

Fig. 2. Overview of chemosensitive factors that influence blood flow,
Detail are presented in the text.

created by the intravascular pressure and the flow-induced
vasodilation due to the wall shear stress exerted by theflowing blood on the endothelium.
The myogenic response
The myogenic response acts in such as way as to tend tomaintain a constant circumferential tension in the arterioles
despite a change in intravascular pressure 11）. A model
description of this mechanism is shown in Figure 3 12）.
Conceptually this mechanism considers the vascular smoothmuscle cell to consist of a contractile element in series with apassively distensible sensor element. It is proposed that as
the sensor element is stretched it causes depolarization of thesmooth muscle cell and excitation of the contractile element.
The length of the sensor element and its degree of
deformation is dependent upon the circumferential tension（T）. Tension is in turn dependent on both the intravascularpressure（P）and the vessel radius（R）, according to the Law
of Laplace T = P・R. Consider now how the arteriole
responds when pressure is elevated. As shown in Figure 3,
an increase in pressure leads to an increase in vessel radiusand deformation of the hypothetical sensor, which in turncauses shortening of the contractile machinery. Note thatwhen the vessel radius returns to its initial level, the wall
tension and sensor length are still elevated due to theincreased pressure. This causes the smooth muscle cell toshorten further until an equilibrium point is reached at which
the sensor is still somewhat deformed at an elevated pressure
and as a consequence, the contractile element remains in ashortened state. While the final details are not clear, there isevidence that integrins, which connect the smooth muscle cell

ARTIFICIAL BLOOD Vol. 14 , No.3, 2006

Fig. 3. Schematic diagram of a model of myogenic regulation ofarteriolar diameter. Details are presented in the text. From（12）by permission.

The myogenic response is found in virtually all thearterioles in different organs and is one of the primarymechanisms that provides a basal level of vascular tone, thatis a partial contraction of the arterioles. Of course theconstant discharge of the sympathetic nervous system fibers
acting on the arteries and arterioles also is important inproviding a continuous state of partial contraction of thesmooth muscle 13）. The functional importance of the myogenic
response relates to the property shown in Figure 3, namelythat it causes vascular tone to be inversely related to
intravascular pressure. Experimental studies have shownthat this mechanism tends to maintain capillary hydrostaticpressure constant as arterial pressure changes. It also tends
to maintain blood flow to an organ constant as arterialpressure changes（autoregulation of blood flow）.
Flow-induced dilation
The second mechanosensitive mechanism, flow-induced
dilation, is a consequence of the wall shear stress dependent
release of nitric oxide（NO）or, in certain vascular beds,
prostaglandins, causing vasodilation14）. This mechanism has
been extensively studied and described elsewhere and will
not be considered in detail here. Flow-induced dilation isfound throughout the arterial and arteriolar networks. It is
functionally important in a number of circumstances. For
example, at the onset of muscular exercise the release ofvasodilator metabolites from parenchymal cells causes
relaxation of the arterioles in the muscle and increases flow
through the arteries upstream that are outside the muscle

81and thus not exposed to the vasodilator metabolites. The
increased flow in these arteries increases wall shear stress on
the endothelium that then leads to NO or prostaglandin
release and dilation.

Influence of the Rheological Properties of Blood
The chemosensitive and mechanosensitive mechanisms
regulating blood flow are influenced by the physicalproperties of the blood, most importantly the viscosity at theinterface between the flow stream and the endothelium since
shear stress in the flow stream is maximal at this interface.
As blood travels through the vessels the red cells tend to
migrate toward the center of the vessel, leaving a cell-freelayer near the vessel wall 15）. The cell-free layer will influence
the resistance to blood flow directly through its viscosity andalso indirectly by influencing the geometric component of theflow resistance through the flow-induced dilation mechanism
described above. We have found that the width of the layer
increases from about 1 μm in arterioles of 10 μm i.d. toabout 3 μm in 50 μm i.d. vessels（unpublished findings）.
As mean width of the cell-free layer increases, the flow
resistance would be reduced since viscosity in the cell-freelayer is lower than in erythrocyte core. On the other hand,
the irregular surface of the interface between the cell-freelayer and erythrocyte core has been shown theoretically toincrease the flow resistance 16）. Secondly, it has been shown intheoretical studies that an increase of the cell-free layer width
would reduce NO scavenging by the red cell core17）, leading
to a higher concentration of NO in the vicinity of the vascularsmooth muscle, which would cause vasodilation. Thisobservation suggests that the width and form of the cell-freelayer near the vascular wall plays a significant role in bloodflow regulation.

Effects of Blood Substitutes on Flow Regulation
The contribution of the cell-free layer to flow regulation
may have significant implications for blood substitutes which
generally consist of a solution containing hemoglobin in someform that increase the volume of the cell-free layer relative tothe red cell component and reduce hematocrit. Since thewidth of the cell-free layer is inversely related to hematocrit,the increased width would reduce the scavenging of NO by
the red cells. However, this effect may be partially offset byincreased scavenging of NO if the blood substitute ishemoglobin-based.
At the systems level it is well known that reducing theoxygen level in the blood by reducing the PO2 of the inspired
gas has profound effects on the cardiovascular system,
causing a reduction in arterial pressure and blood flow, 18）.

82

These changes accentuate the local effects of the hypoxemia
itself. It seems likely that blood substitutes would also
introduce systemic as well as local effects.
From the considerations presented above, it is evident thatthe physiological effects of blood substitutes go well beyond
those due to simply restoring the oxygen carrying capability.
As a consequence, in designing a blood substitute its effects
on the local regulatory mechanisms and the stimuli to which
they are sensitive must be carefully considered.

Summary
In summary, blood flow to individual organs is determinedlargely by local regulatory mechanisms that respond to avariety of influences at the level of individual arterioles.
These mechanisms fall generally into two main categories,
chemosensitive and mechanosensitive. Chemosensitive
mechanisms respond to changes in the environment of thearteriole and of the parenchymal cells as determined byoxygen levels in these regions. Mechanosensitive mechanisms
respond to wall shear stress produced by the cell-free layerof the flow stream acting on the endothelium andcircumferential stress acting on the vascular smooth muscle.
Blood flow through each arteriole is determined by thesummated effect of the stimuli presented to the arteriole bythese operative mechanisms. In turn the organ flow
represents the net effect of these independent processes
occurring in individual arterioles.
Cardiac output then represents the summation of literally
millions of the individual processes within each organ. In
other words, cardiac output in the steady state situation isdetermined by the demand of the individual organs and notby the command of central mechanisms. When blood
substitutes are introduced into the circulation local control
mechanisms are significantly affected through changes inoxygen delivery, intravascular pressure, blood viscosity, wall
shear stress and NO scavenging by the blood substitute andthe red blood cells. A better understanding of the effects ofblood substitutes on the chemo- and mechanosensitive factorsthat control blood flow through their effects on the arteriole
may aid in developing more effective blood replacements.

Acknowledgements:
The excellent technical assistance of Christina Yee-Collins,
Kristina Flores and Scott Dunning is gratefully
acknowledged. Supported by NIH grants HL52684 andHL66318 to P.C.J and HL62318 to M.I.

References
１. Johnson PC. Review of previous studies and current

人工血液 Vol. 14 , No.3, 2006

theories of autoregulation. Circulation Res. 1964;15（Suppl.
1）: 2-9.
２. Broten TP. and Feigl EO. Role of myocardial oxygen andcarbon dioxide in coronary autoregulation Am J Physiol
1992;262: H1231-7.
３. Kontos HA, Wei EP, Raper AJ and Patterson JL Jr. Local
mechanism of CO 2 action of cat pial arterioles: Stroke
1977;8:226-29.
４. Haddy FJ and Scott JB. Metabolic factors in peripheral
circulatory regulation. Fed. Proc. 1975;34:2006- 11.
５. Shubert RW, Whalen WJ, and Nair P. Myocardial PO2
distribution, relationship to coronary autoregulation. Am J
Physiol 1978;234: H361-H370.
６. Toth A, Pal M, Tischler ME and Johnson PC. Are there
oxygen-deficient regions in resting skeletal muscle? Am J
Physiol 1996;270: H1933-9.
７. Pal M, Toth A, Ping P and Johnson PC. Capillary bloodflow and tissue metabolism in skeletal muscle during
sympathetic trunk stimulation. Am J Physiol 1998;274:
H430-440.
８. Prewitt RL and Johnson PC. The effect of oxygen on
arteriolar red cell velocity and capillary density in the rat
cremaster muscle. Microvasc Res 1976;12: 59-70.
９. Johnson PC. Chapter 9.Local regulation of blood flow.
In:Blood Substitutes, R.M. Winslow, Ed. Academic Press.
Amsterdam. 2006;102-111.
10. Hester RL and Hammer LW. Venular-arteriolar
communication in the regulation of blood flow Am J
Physiol Regul Integr Comp Physiol 2002;282: R1280-5.

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11. Johnson P. The Myogenic Response（1980）In Handbook of
Physiology Section 2 The Cardiovascular System. Volume
II: Vascular Smooth Muscle（Ed: Bohr DF, Somlyo AP,
Sparks HV）American Physiological Society, Bethesda,
1980;409-442.
12. The microcirculation and local and humoral control of thecirculation. In: Cardiovascular Physiology,（Ed: Guyton AC
and Jones CE）Baltimore, Univ. Park, 1974;163-195.（Intern.
Rev. Physiol.Ser. vol 1）
13. Sparks H. Chapter 7 Skin and Muscle In Peripheral
Circulation（Ed, Johnson P）John Wiley and Sons, Inc.,New York, 1978;193-230.
14. Smiesko V and Johnson PC, The Arterial Lumen isControlled by Flow-Related Shear Stress. News Physiol
Sci 1993;8: 34 - 38.
15. Kim S, Kong RL, Popel AS, Intaglietta M and Johnson PC,
A computer-based method for determination of the cellfree layer width in microcirculation. Microcirculation
2006;13:199-207.
16. Sharan M and Popel AS. A two-phase model for flow ofblood in narrow tubes with increased effective viscosity
near the wall. Biorheology 2001;38: 415-28.
17. Vaughn MW, Kuo L and Liao JC. Effective diffusion
distance of nitric oxide in the microcirculation. Am J
Physiol 1998;274: H1705-14.
18. Johnson PC, Vandegriff K, Tsai AG, and Intaglietta M.
Effect of acute hypoxia on microcirculatory and tissue
oxygen levels in rat cremaster muscle. J. Appl. Physiol
2005;98: 1177-84.

83

Review

Polyethylene Glycol Conjugated Albumin:
A New Generation Plasma Expander
Nanae Hangai-Hoger and Marcos Intaglietta

Abstract
Polyethylene glycol conjugated albumin（PEG-Alb）in either normal saline or Ringer's lactate at concentrations in the ranging
from 2.5 to 4.0 g/dl is a non-immunologic, non-antigenic plasma expander that has a long intravascular permanence andmaintains central blood pressure, blood flow, and functional capillary density in extreme hemodilution, hemorrhagic shock andendotoxemia. It has longer lasting effects（half life of the order of 24 hours）and is more efficacious than colloids such as starch,albumin and dextrans and has the specific of restoring and improving microvascular function. These properties suggest that its
use in critical illness and resuscitation can delay the use of blood transfusions, thus extending the transfusion trigger to lower
hemoglobin levels.
Keywords

PEG-Albumin, plasma expanders, shock, extreme hemodilution, endotoxemia, functional capillary density

INTRODUCTION
The need for an optimal plasma expander is as essential asthat for a blood substitute. Factors that define an optimal
plasma expander are the ability to sustain volume expansion
for a prolonged period, maintenance of central blood pressure,lack of red blood cell（RBC）aggregation, effectiveness at low
concentration and maintenance of tissue perfusion. How toachieve these properties has been a subject of controversy
particularly regarding the viscosity, colloid osmotic pressure（COP）and type of material needed to obtain properties that
insure adequate organ perfusion. Maintenance of tissue
perfusion is probably the most important parameter inclassifying the effectiveness of a plasma expander. Tissue
blood flow is determined by perfusion pressure, vascular
resistance and blood viscosity, factors that in rigid tubes are
related by Poiseuille's equation. However, the introduction ofplasma expander may lead to in vivo changes in oncotic andosmotic pressure and blood viscosity that interact withregulatory processes of peripheral vascular resistance arisingfrom changes in the production of vasoactive mediators by
the endothelium and their transport in the circulation.
Introduction of a plasma expander invariably reduces
hematocrit（Hct）, and unless its fluid viscosity is similar tothat of blood it reduces blood viscosity. The reduction ofblood viscosity（hemodilution）has been regarded as beneficial
since antiquity. Currently the limit of hemodilution is thecondition when perfusion and oxygen delivery are no longer
able to maintain tissue metabolism, a point termed thetransfusion trigger, where restoration of blood oxygen
carrying capacity is considered necessary. Studies by Tsai etal. show that as blood viscosity is reduced by hemodilution,
microvascular function is progressively impaired,jeopardizing tissue survival due to the local microscopic
maldistribution of blood flow 1）. These effects take place at
Hcts that are greater than those defining the oxygen supply
limitation, a finding that leads to the hypothesis that the limitof hemodilution could be significantly decreased if the processof plasma expansion maintains microvascular function.
Recent studies show that microvascular function can be
maintained in extreme hemodilution by increasing either
blood or plasma viscosity 1-3）. Restoration of blood viscosity
during hemodilution and hemorrhage is desirable, because it
maintains functional capillary density（FCD）, defined as thenumber of capillaries with passage of RBCs per unit surfaceof the field of view of a microscopically observed tissue. This
microvascular parameter was found to be critical in defining
tissue survival by Kerger et al. 4）, who showed the direct
correlation between maintenance of FCD above a specific

Department of Bioengineering University of California, San Diego 9500 Gilman Dr. Mail code 0412 La Jolla, CA 92093-0412
論文受付 2006年６月23日 論文受理 2006年12月11日

84

人工血液 Vol. 14 , No.3, 2006

threshold and survival in extended hemorrhagic shock 4）. FCD
is also determined by the maintenance of capillary pressure,which in extreme hemodilution is obtained by using highviscosity plasma expanders 1）.
The blood viscosity threshold that causes the decrease in
FCD appears to coincide with the decision of transfusing
blood. Therefore, the transfusion trigger may also be a"viscosity" trigger, and some of the results obtained with ablood transfusion may also be achieved by increasing plasma
viscosity. Thus use of RBCs for the purpose of increasing
blood viscosity is unnecessary if a material is introduced that
increases plasma viscosity in the circulation. In this context it
would seem that a desirable property for plasma expanders
is that of increasing blood viscosity.

The viscosity of plasma expanders
The viscosity of plasma expander is determined principallyby the size of the colloidal component and its concentration.
Historical non-crystalloid plasma expanders were formulated
to limit their viscosities. Gelatin, dextran and starch solutions
were formulated in concentrations leading to viscosities in therange of 2 cp, prior to administration. The inherent dilution of
the material upon their introduction into the circulation
determine that final plasma viscosity was not very different
from normal, i.e., about 1.0 to 1.2 cp. The viscosity of acolloidal solution is determined by both the number ofparticles per unit volume, and the molecular volume of thesolute. Therefore augmenting concentration is not amechanism for increasing viscosity of plasma expanders since
this increases COP, bringing interstitial fluid into thecirculation, diluting the material thus lowering viscosity, aself-limiting process.
A different approach for increasing solution viscosity is toincrease molecular dimensions. Several molecular species
have been proposed and tested as a basis for plasma
expanders, including poly-vinyl pirrolidone（PVP）, highmolecular weight dextrans and starches, keratins, alginatesand polyethylene glycol conjugated albumin（PEG-Alb）.
With few exceptions high molecular weight material suchas PVP, starches, dextrans and keratins tend to cause RBC
aggregation when Hct is near normal. Alginates and PEG-Alb
do not cause RBC aggregation and have similar properties
since both materials trap a large amount of water in theirmolecular structure, which causes the increase of theireffective dimensions. These materials have very different
plasma expansion characteristics since alginates have COP of

ARTIFICIAL BLOOD Vol. 14 , No.3, 2006

2 - 3 mmHg when used at physiological concentrations（in therange of 0.7 - 0.8% by weight）while current formulations of
PEG-Alb at about 4% concentration have COP of about 60
mmHg. At present alginates have not been extensively
studied, while there is substantial information on thephysiological characteristics on plasma expansion by PEGAlb, also because it has the same biophysical properties as
PEG-Hemoglobin, a material presently in clinical trials as anoxygen carrying blood substitute.

PEG-Albumin formulation and properties
PEG-Alb is formulated with either bovine or humanalbumin using similar procedures. Polyethylene glycol（PEG）
has been used for modifying of proteins, peptides, enzymes
and liposomes to extend plasma half-life, eliminate/lower
toxicity, immunologic reactions and antigenicity, increasesolubility in water and provide increased thermostability 5-9）.
PEG-Alb is produced by conjugating the protein with PEG
using a single step version of the thiolation mediated,maleimide chemistry based conservative PEGylation
described by Acharya et. al., 10）. Albumin（0.25 mM）isincubated with 5 mM 2-iminothiolane（BioAffinity Systems,
Rockford, IL）with 7.5 mM maleimide phenyl PEG-5,000 in
PBS overnight. The surface amino groups are thiolated andthiol groups generated on the protein in situ are derivatizedby the maleimide PEG in the reaction mixture. The single
step reaction used limits the oxidation of the thiols of thethiolated protein, and is a general approach for producing
PEGylated proteins. Excess reagents are removed bytangential flow filtration after the overnight incubation and a
70 K membrane is used for diafiltartion and removal of
unreacted PEG and excess immunothiolane. The material isprepared in concentration ranging from 2 to 4 g/dl（protein
based）. This chemical modification leads a colloidal osmoticpressure 40 mmHg, and a viscosity of 2 cp for a 2.5 g/dl
concentration in saline.
The PEG molecule has the property of trapping asignificant amount of water in its vicinity and a few PEG
polymers attached to the surface of a protein trap a layer ofwater on it surface. This process causes the hydrodynamic
dimensions of the protein to increase substantiallyaugmenting its intrinsic viscosity and the COP of the solution.
Biologically the water layer renders the molecule
undetectable by receptors and the immune system which
endows PEG treated molecules with "stealth" or"immunosilent" properties.

85

PEG-Alb is usually formulated with an average of 6-12
copies of PEG 5 kDa resulting in a molecular radius of theorder of 8-9 nm vs. a radius of 2.5 nm for the natural albumin
molecule. Such a large radius would cause a significant
increase in viscosity, however the viscogenic properties arecomparatively moderate, and changes in viscosity are notmaintained when the material is introduced into thecirculation because the high COP dilutes PEG-Alb to theextent that plasma concentrations are seldom beyond 1%,
leading to plasma viscosities of 1.3 cp vs. the normal plasma
viscosity of 1.2 cp.
At present there are no known biological or functional
differences between PEG-Alb made with human, bovine orrecombinant albumin, also because the PEG conjugation
isolates the protein from the immune system.

improved for PEG-Alb since flow was normalized and FCD
was above 60%, but not for Dex 70 and HSA whose flows
were significantly below normal and FCD was below 50%.
These results should be in part due to the normalized
oxygen delivery to the heart, since the microvascularconditions found in the window chamber tissue should becommon to those of other tissues, as shown by a recent studyof where organ flow distribution was measured 12） in extreme
hemodilution. The results obtained appear to be primarily
due to vasodilatation induced by PEG-Alb, while the opposite
effect was seen for Dex 70 and HSA as shown in Figure 1.
Vasodilatation and the significant decrease in peripheral
vascular resistance may be also the reason why mean arterial
blood pressure did not attain baseline values.

PEG-albumin in extreme hemodilution
Extreme hemodilution is a condition that may be defined as
the dilution of blood with plasma expanders beyond thetransfusion trigger. It is seldom attained in clinical conditions
unless continued blood losses are corrected with plasma
expanders in the attempt to manage blood volume whenblood is not available. Physiologically it is defined by theblood oxygen carrying capacity beyond which oxygen
consumption becomes dependant on hemoglobin
concentration. This critical condition provides an important
experimental benchmark for studying the efficacy of blood
substitutes, because changes in the circulating blood
properties due to the presence of a test material clearly
defines its effectiveness in either sustaining, improving or
deteriorating systemic and microvascular parameters over a
very narrow range of changes in blood composition. This
acute anemic condition magnifies the effects of test
compound since in this condition the organism is only
marginally capable of compensating for changes in blood
properties.
Cabrales et al., made extreme hemodilution experiments via
a two step procedure with 6% dextran 70（Dex70）to a Hct〜
18% and then hemodiluting to Hct 11% using 5% humanserum albumin（HSA）, 4% PEG-Alb（HSA）（MPA）, Mal-PEGhemoglobin,（MP4, P50 = 5.4 mmHg, Sangart Inc., San Diego,CA）and 6% Dex 70 11）. Systemic findings were that PEG-Alb
provided a greater blood pressure than both HSA and Dex
70, and increased cardiac index 44% above baseline（nohemodilution）. Notably systemic peripheral vascular
resistance for PEG-Alb was 70% of that for the non PEG
materials. Microvascular conditions were significantly

86

Fig. 1. Extreme hemodilution to Hct 11% with 4.2% PEG-Alb compared
to 6% Dextran 70 kDa（Dex 70）and 5% human serum albumin
HAS. Vessel diameter, functional capillary density and cardiac
index after level 3 exchange are shown relative to baseline. There
was no statically significant difference in plasma and blood
viscosity between groups. Plasma COP was 21.4 ± 0.9 mmHg for
PEG-Alb, 20.3 ± 1.1 mmHg for HAS, and 16.8 ± 0.8 for Dex 70.
P< 0.05 vs. baseline（*）, P<0.05 vs. Dextran 70（†）. Data from
Cabrales et al., 2005.

PEG-Alb produces two vasodilatory effects, one radicatedin the arteriolar and venular circulation and a second
manifested by the increase in FCD. The former is clearly aconsequence of a vasoactive phenomenon, since it is related
to the reactivity of smooth muscle, which PEG-Alb causes torelax. The increase or maintenance of FCD is passive effectdue to the transmission of blood pressure to the capillary
network, which follows from the absence of vasoconstrictionin the arteriolar circulation. A likely mechanism behind thevasodilatory effect of PEG-Alb should be NO management by
PEG-Alb.

人工血液 Vol. 14 , No.3, 2006

Nitric oxide mechanism and PEG-Alb
A mechanism that could in principle account for avasodilatory effect is mechano transduction resulting fromthe maintenance or restoration of shear stress on theendothelium. In extreme hemodilution where PEG-Alb
appears to be effective in maintaining flow and microvascularfunction shear stress is low due to the significant decrease inblood viscosity and the prevailing low flow conditions present
with the use of low viscosity plasma expanders. Shear stressis a mediator of the production of NO by mechanotransduction by the endothelium13，14）, therefore increasing flow
and viscosity should increase production of NO by theendothelium, an effect that was demonstrated by Tsai et al.,using the high viscosity plasma expander Dextran 500 kDa.
This effect has increased effectiveness in extreme
hemodilution because the significant decrease in hematocrit
lowers the NO scavenging properties due to the decrease of
total hemoglobin in blood, near the source of production in
the endothelial layer 15）.
The mechano transduction hypothesis has been
demonstrated for high viscosity plasma expanders such a
dextran 500 kDa in extreme hemodilution 1）, alginates inhemorrhagic shock 16）, and the use viscogenic RBCs that do
not carry oxygen（hemoglobin converted to CO-hemoglobin）
in hemorrhagic shock, and therefore achieve resuscitation
solely by virtue of their enhancement of blood viscosity 17）.
However, the viscogenic properties of PEG-Alb are notsufficient to increase plasma viscosity significantly, also
because the high COP dilutes the protein to the point that it
is seldom possible to attain plasma concentrations greaterthan 2 g/dl. In summary, the mechano transduction
hypothesis works when viscosity can be increased without
increasing COP, which is not the case for PEG-Alb.
PEG-Alb appears to be unique in causing vasodilatation in
conditions of anemia, low viscosity, and low oxygen tension. It
is significant that the chemical process that leads topegylation of albumin requires the thiolation of the surface
amino groups leading to the production of extra thiol groups
on the protein surface. Thiol groups have been implicatedwith the transport of NO from high concentration regions of
the circulation, like the aortic wall, to the lower NO
concentration regions of the microcirculation18）. In this process
PEG-Alb could provide a source of extra thiols if the chemical
process of pegylation does not neutralize all active sites
generated on the albumin by thiolation. These mechanisms
have been proposed to occur in normal blood and to be a partof the regulatory control of ischemia via dilatation and

ARTIFICIAL BLOOD Vol. 14 , No.3, 2006

increased perfusion 3）. According to this mechanism, the sameprocess may be enhanced by the presence of unreacted thiols
on the surface of PEG-Alb. Therefore the unusual
vasodilatory capacity of PEG-proteins the enhancement of an
NO transporting mechanism may be due to pegylation via
thiolation.

PEG-albumin in hemorrhagic shock
Hemorrhagic shock results from the loss of circulatory
volume and oxygen carrying capacity, causing increased
heart rate, vasoconstriction, redistribution of blood flow awayfrom nonvital organs and the decrease in capillary perfusions
and FCD. Restoration of lost volume with plasma expanders
is the initial therapy followed by blood transfusion.
Cabrales et al. compared the plasma expander HES 200（Hydroxyl ethyl starch, Pentaspan, B. Braun, Medical , Irvine
CA, 10% w/v COP 85 mmHg, viscosity 3.4 cp）with PEG-Alb（2.5 g/dl, COP 38 mmHg viscosity 3.4 cp）in a conventional
shock protocol where 50% of blood volume is removed in 5
min, and 25% of blood volume is returned duringresuscitation 19）. This study showed that PEG-Alb and HES
provide initial identical systemic and microvascular recovery
for about 15 min after resuscitation, when a trend started
leading to statistically significant higher flow, mean arterial
blood pressure, FCD and tissue pH for PEG-Alb in 30 min（Figure 2）. Mechanistically the difference in recovery was
related to the of lack of sustained recovery of arteriolar flow
and FCD hindering the washout of metabolites from thetissue, preventing lactic acid from returning to normal levels
causing the incomplete restoration of positive base excess
and limiting the normalization of pH. The improved
resuscitation found with PEG-Alb vs. HES should also be due
to effects in the heart muscle and sustained improved cardiac
function, as evidenced by the extended maintenance of bloodpressure and improvement of arteriolar flow.
The difference in outcome could be due to the significant
longer retention time of PEG-Alb a monodisperse material
with molecular weight 130 kDa, vs. HES broad spectrum of
molecular weights with a significant portion of material < 200
kDa. An alternative and/or complimentary explanation forthese effects is that PEG-Alb caused a thiol mediatedimproved distribution of NO, leading to vasodilatation,
improved flow and sustained cardiac function and bloodpressure, as seen in extreme hemodilution, since there was nodifference in blood viscosity, and plasma viscosity was 1.4 cp
which is below the level needed to sustain FCD ranging from
1.8 cp 17）to 2.2 cp 1）.

87

Fig. 2. Hemorrhagic shock resuscitation with 2.5% PEG-Alb vs. 10% hydroxyl ethyl starch, HES 200（Pentaspan, B. Braun, Medical , Irvine CA）.
Systemic and microvascular parameters were maintained by PEG-Alb through the observation period. HES produced an initial recovery thatwas identical to PEG-Alb, but this was not sustained beyond the initial 15 min resuscitation period. Microvascular parameters diverged ahead tosystemic parameters by 5 minutes after resuscitation. P<0.05 between HES and PEG-Alb（†）. Data from Cabrales et al., 2005, reprinted bypermission.

The significance of restoring oxygen carrying capacity inresuscitation from hemorrhagic shock was illustrated in the
study of Wettstein et al. who used the conventional shockmodel resuscitated with PEG-Alb only and PEG-Alb andRBCs up to a concentration of 8 g/dl hemoglobin in theresuscitation fluid 20）. In this study, microvascular blood flow
and FCD was significantly improved by avoiding thetransfusion of additional RBCs while increasing the fraction of
PEG-Alb solution in the resuscitation fluid. However, thehighest oxygen delivery was obtained using PEG-Alb with 8
g/dl of hemoglobin in RBCs. As expected animals receiving
RBC transfusion had higher oxygen tensions, however
resuscitation without additional RBCs extracted a higher
percentage of the available oxygen. A conclusion from thisstudy is that microvascular recovery is a strong function of
the biophysical properties of the plasma expander, andindependent from the restoration of oxygen carrying
capacity.

88

The relevance of oxygen carrying capacity in shock
resuscitation was also tested by Wettstein et al., in thestandard shock model using PEG-Alb and an identicallyconfigured PEG-Hemoglobin（PEG-Hb）, where the latter is amolecule with a high affinity for oxygen, having a p50 of 5.4
mmHg 21）. These molecules, which are biophysically identicalwere used in the same concentration（4.2 g protein/dl）, whichwas diluted to about 1 g/dl upon introduction into thecirculation. This study showed that volume restitution in thehamster window chamber model of hemorrhagic shock did
not show any microvascular or systemic functional
differences between molecules, with identical recovery ofsystemic blood pressure, acid base balance and FCD. As
expected oxygen delivery and consumption by PEG-Alb wassignificantly lower than that obtained with PEG-Hb, andtissue pO 2 was also significantly lower. Therefore thiscomparison further supports the concept that thecharacteristics of the plasma expander are critical for therecovery of hemodynamic parameters, which is not

人工血液 Vol. 14 , No.3, 2006

necessarily related to the restoration of oxygen carrying
capacity.

PEG-Albumin for the treatment of sepsis
Sepsis is an inflammatory response that produces
proteolytic enzymes and oxygen metabolites that cause tissue
damage. This damage is prevented by antioxidants, capableof blocking or inactivating the noxious products ofinflammation. Sepsis can also be accompanied by hypotension,a condition called septic shock where, prior to the systemic
circulatory collapsed there is wide spread microcirculatory
impairment and tissue hypoxia leading to organ failure. The
conventional therapeutic approach based on the VIP principle
22）（ventilate, infuse, and pump）
is deployed in the early stageof sepsis.
We studied the effects infusing a PEG-Alb solution
administered as 30 % of blood volume in a hypervolemicbolus in the hamster chamber window model of LPS induced
endotoxemia 23）. This model showed a significantly decreased
FCD（less than 10% from baseline）at 6 hrs after LPS
injection, which did not recover during observation period（24hrs）without treatment. Fluid resuscitation was begun inearly stage of endotoxemia as recommended using PEG-Alb
infused with either 16 ml/kg（PEG-Alb-16）or 24 ml/kg（PEGAlb-24）concentration intravenously during 1 hour, andcompared this to Dex 70（6% wt/vol molecular weight 70 kDa
B. Braun Medical, Irvine, CA）infused at 24 ml/kg hr.
This study showed that treatment with PEG-Alb restored
impaired microvascular function by increasing FCD andtissue pO2 to near normal levels, while lowering perivascular
NO concentration when compared to treatment using
dextran 70 kDa. Endotoxemia significantly increased
perivascular NO in arterioles and venules, which was also
reduced by the increased perfusion due to PEG-Alb-24
treatment. There was also a significant decrease of
perivascular tissue pO 2 at all time points of observation,
which was in part corrected by the increased perfusion due
to PEG-Alb-24, but not by dextran treatment. In a separate
study recombinant human serum albumin（Nipro Medical
Corp., Osaka, Japan）was used in the same protocol, which
provided a similar recovery of FCD at 6 hrs after LPS as,
PEG-Alb, however the latter had significantly better outcome
24）
after 12 hrs（Figure 3）.

ARTIFICIAL BLOOD Vol. 14 , No.3, 2006

Fig. 3. Treatment of endotoxemia. No treatment（NR）reduced functional
capillary density（FCD）to below 10% at 6 hr point, and remained
below 20% of baseline for a 24 hr. period. Fluid resuscitationconsisted in the infusion of 24 ml/kg/hr for one hour of 6%
Dextran 70 kDa（Dex70）or 8.3% recombinant HAS（rHSA）, and
2.5% PEG-Alb（PEG-Alb）. Resuscitation with Dex 70 increased
FCD, only at 2 hrs. rHSA increased FCD, which reached amaximum at 12 hrs and remained at this level until the end ofobservation period. PEG-Alb improved FCD gradually until theend of the experiment. P<0.05 vs. baseline（*）; P<0.05 vs. NR（†）;
P<0.05 vs. Dex70（‡）;p<0.05 vs. rHSA（§）. Data in part reported
by Hangai-Hoger et al., 2006.

Microcirculatory impairment in sepsis is correlated withincreased tissue NO concentration induced by iNOS 23，25）. NO
is known to have a dual personality 26）whereby NO induced
by eNOS at physiological levels has beneficial circulatory
effects, while NO induced by iNOS causes circulatory
impairment, its level being 100-1000 times higher which ispathological. In the hamster model perivascular NO
concentration was increased to mM level（nM being normal）
and PEG-Alb resuscitation decreased NO to normal levels, aneffect probably resulting from the combined action of PEGAlb maintain microcirculatory flow and FCD, increasing theavailability of RBC hemoglobin for scavenging NO. Likewise
the mechanism of NO thiol transport may also have been
operational, in a reverse mode, whereby NO is uploaded in
the microcirculation and distributed to organs with lower
iNOS activity.

CONCLUSIONS
Fluid therapy in critical care is still a matter ofcontroversy. Colloidal solutions have advantages overcrystalloids because they are effective in lower volumes.
However, colloids are expensive, present the risk of allergic
reaction and coagulopathic effects. Notably PEG conjugationof colloids should reduce this potential toxicity while also
reducing the total amount of protein administered.

89

PEG conjugated colloids particularly albumin andhemoglobin have excellent plasma expansion properties,
thorough effects that are not explained by their biophysicalproperties, also because these materials have high COPs
causing their concentration in the circulation in mostresuscitation scenario to be too small to affect blood viscosity,
shear stress and mechano transduction. Analysis of theavailable data shows that both PEG-Alb and PEG-Hb cause
vasodilatation in extreme hemodilution, which in the absenceof mechano transduction would appear to be mediated via
NO transport.
An important feature of PEG-colloids is that they havelarge hydrodynamic radii increasing their intravascularretention time, since they cannot be filtered into the tissues.
Even though it would be in principle possible to simply
provide albumin with extra thiols to obtain the NO mediatedvasodilatation, this materials would extravasate according to
the exchange transport dynamics of natural albumin which
has a significantly lower intravascular residence time. In
principle any molecular species of sufficient size including
dextrans and starches could serve as a frame to support PEG
molecules, however albumin（and hemoglobin）have thedesirable feature of being uniform in dimensions with apredictable number of surface molecular structure forattaching PEG via thiolation.
It is not clear at this time what will be the economic
scenario for PEG-Alb, however it is likely that it will be atleast as costly as the basic colloid. This problem however
should be compensated by its unique volume expansion andmicrovascular function restoration and enhancement
properties, coupled with its effectiveness at weight
concentrations that are significantly smaller than theunmodified colloid. An additional economy inherent to the useof this new type of plasma expander is that its ability ofmaintaining microvascular function allows extending the RBC
transfusion trigger.
It is likely that PEG-HSA will be effective in extending thetransfusion trigger; however an oxygen carrier is ultimately
required when blood losses are extensive. Therefore it may
be effective to consider the use of a plasma expender like
PEG-Alb in combination with a material such as HbV（hemoglobin vesicles）which carries oxygen but like RBCs
has no COP per se, and requires an appropriate fluid vehicle.

ACKNOWLEDGEMENTS
This research was conducted with the financial support

90

from the USPHS grant Bioengineering Research Partnership
R24-HL64395 and grants R01-HL62354 and R01-HL62318 toMI. M. Intaglietta is a member of the Board of Directors of
the La Jolla Bioengineering Institute.

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５. Kawahara NY, Ohno H. Induced thermostability of poly
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16. Cabrales P, Tsai AG, Intaglietta M. Hyperosmotichyperoncotic vs. hyperosmotic-hyperviscous small volume
resuscitation in hemorrhagic shock. Shock 2004;22:431-437.
17. Cabrales P, Intaglietta M, Tsai AG. Increase plasma
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Review

Hemoglobin Vesicle Aids Recovery of Cardiac Function during
Ischemia-Reperfusion in Langendorff Perfused Rat Hearts
Tadashi Yamagishi, Motoaki Bessho, Miyuki Hama, Ryuichi Katoh,
Shigeki Yanagida, Masatoshi Kusuhara, Fumitaka Ohsuzu

Abstract
Background. - Formed from a lipid bilayer membrane, hemoglobin vesicle（HbV）is a small sphere（diameter ca. 250 nm）whichcontains hemoglobin, and is a candidate blood substitute. In this study, we examined whether HbV influences cardiac function
during ischemia-reperfusion.
Methods. - Rat hearts were perfused according to the Langendorff method and subjected to 30 min of global ischemia and 30
min of reperfusion. HbV was made into a dispersion and diluted with Krebs-Henseleit buffer to achieve hemoglobin
concentrations of 0.33 g/dL and 0.10 g/dL, and the hearts were perfused with this dispersion for 10 min immediately prior toischemia. The same experiment was performed using the empty vesicle（EV; no hemoglobin）. Cardiac functions were
continuously monitored and coronary effluent collected every 5 min throughout the experiment.
Results. - In the HbV groups, between 10 and 30 min of reperfusion, there was a significant recovery in heart rate（to virtually
pre-ischemia levels）as compared to the control group（p<0.05）. A rise in endodiastolic pressure during reperfusion was
significantly suppressed in the HbV group（30-60 mmHg）as compared to the control group（70-100 mmHg）（p<0.05）. In the HbVgroups, there was a significant recovery in left ventricular developed pressure（LVDP）between 20 and 30 min of reperfusion,as compared to the control group（p<0.05）. After 5 min of HbV perfusion and at 1 min of reperfusion, the lactate concentrationin the coronary effluent was significantly lower in the HbV 0.33 g/dl group than in the control group（p<0.05）.
Conclusion. - These results suggest that HbV changed cardiac metabolism before and during ischemia, and as a result,enhanced recovery of cardiac function during reperfusion.
Keywords

hemoglobin-vesicle, cardiac functions, lactate, ischemia-reperfusion

Introduction
Artificial red blood cells have the following features that
reduce problems in clinical blood transfusions:（1）no need fortime consuming cross-matching or typing,（2）no need forrefrigeration, and（3）no potential as infectious agents.
Therefore, artificial red blood cell substances have been
widely investigated as potentially useful blood substitutes
since the 1960s1）. They include perfluorochemicals, various
types of chemically modified hemoglobin, and recombinanthuman hemoglobin2-3）, and are more simple oxygen-carriers
than artificial red blood cells. In 1993, Takeoka et al. 4）
developed a method for the production of hemoglobin vesicles
covered with a lipid bilayer membrane that could serve as
artificial red blood cells. Since then, many studies on thefunctions5-6）of hemoglobin vesicles as a blood substitute andtheir biological safety7）have been conducted using various
animal models. The data obtained in these studies suggested
that hemoglobin vesicles had no serious adverse effects on
cardiac functions in the models in which they were used but
their actual effects on cardiac functions were not clarified.
Therefore, in the present study, we examined whether HbV
influences cardiac functions during ischemia-reperfusion
using isolated rat hearts and the Langendorff perfusion
technique. We found that HbV aids recovery of cardiac
functions during ischemia-reperfusion in this model.

Materials and Methods
Hemoglobin-vesicle and empty-vesicle（diameter ca.
Hemoglobin-vesicle（HbV）, a small sphere8）
250 nm）formed from a lipid bilayer membrane which

Internal Medicine I, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan
論文受付 2006年10月13日 論文受理 2006年12月４日

92

人工血液 Vol. 14 , No.3, 2006

contains hemoglobin, is supplied by Oxygenix Co., Ltd. as asaline-suspension containing 10 g/dL of hemoglobin. Also
supplied as a suspension, empty-vesicle（EV）is identical to
HbV except that it does not contain hemoglobin. The HbV
suspension was diluted with modified Krebs-Henseleit buffer
to final hemoglobin concentrations of 0.33 g/dL（30 times
dilution）and 0.10 g/dL（100 times dilution）. The EVsuspension was diluted in the same manner as HbV withmodified Krebs-Henseleit buffer.
Animals and experimental groups
Eight-week-old male Wistar rats were purchased fromCharles River Japan Inc. The rats were maintained under
specific pathogen-free conditions and a constant dark/lightcycle（12 h each）in our animal facility at the National
Defense Medical College throughout the experiment. They
were given free access to a laboratory chow CE-7（CleaJapan, Tokyo）and water for a few weeks after purchase. A
total of 33 rats were included in the present study and theexperiments were performed when they were 9 to 12 weeks
old. They were divided into five experimental groups: control
group（n = 6）
, hemoglobin-vesicle 0.33 g/dL group（HbV 0.33
g/dL group, n = 6）
, hemoglobin-vesicle 0.10 g/dL group（HbV
0.10 g/dL group, n = 7）
, empty-vesicle 0.33 g/dL group（EV0.33 g/dL group, n = 7）
, and empty-vesicle 0.10 g/dL group（EV 0.10 g/dL group, n = 7）
. All experiments were
performed in accordance with the National Defense Medical
College Institutional Animal Care and Use Committee Guidelines.
Heart preparation and perfusion method
The rats were pre-medicated with heparin（1,000U, i.p.）,
and 10 min later anesthetized with ketamine hydrochloride（90 mg/kg, i.p.）and xylazine hydrochloride（10 mg/kg, i.p.）.
The hearts were excised, put into ice-cold modified KrebsHenseleit buffer（mKH buffer）, quickly trimmed, weighed,and perfused according to the Langendorff mode（Fig. 1.）.
Perfusion was conducted at a constant perfusion pressure of
100 cmH2O at 37°
C with modified Krebs-Henseleit（mKH）
buffer solution, which comprised NaCl 116 mM, KCl 4.7 mM,
MgSO4 1.2 mM, CaCl2 2.5 mM, NaHCO3 25 mM, KH2PO4 1.2
mM, and glucose 11 mM. The experimental buffer solutions
used（mKH buffer solution, two HbV containing mKH buffer
solutions, two EV containing mKH buffer solutions）were
continuously aerated with 95% O2 + 5% CO2 and the pH wasadjusted to 7.4. Cardiac function was monitored and recorded
using a fluid-filled left ventricular balloon in line with atransducer（P-50, Gould Inc.）and a WS-641G multi-channel
recorder（Nihon Kohden, Tokyo, Japan）. The balloon volume
was set to produce a left ventricular end-diastolic pressure

ARTIFICIAL BLOOD Vol. 14 , No.3, 2006

（LVEDP）of 0-5 mmHg.
In the control group, each heart was perfused with mKH
buffer for 20 min（control perfusion）and then subjected to 30
min of global ischemia by stopping the perfusion. This wasfollowed by 30 min of reperfusion. In the other experimentalgroups, each heart was subjected to perfusion with mKH
buffer for first 10 min of the control perfusion and thenperfusion with the respective vesicle containing buffer
solution for the remaining 10 min. They were then subjected
to 30 min of global ischemia and 30 min of reperfusion in thesame manner as for the control group. To measure thecoronary flow rate（CFR）and lactate content, the coronary
effluent was collected at 5-min intervals during the control
perfusion. During reperfusion, it was collected at 1 min and 4min, and then at 5-min intervals until the end of experiment.
After measuring the volume of the effluent, part of it wascentrifuged at 10,000 x g for 40 min, and the supernatant wasfrozen and stored at -80°
C until analysis for lactate content.

Fig. 1. Schematic presentation of Langendorff perfusion

Biochemical analysis and analysis of results
The coronary effluent was analyzed for lactate content
enzymatically by the method of Lowry and Passonneau9）.
Cardiac function data［heart rate（HR）, left ventricularendodiastolic pressure（LVEDP）, and left ventriculardeveloped pressure（LVDP）］were taken from the records
made at 5-min intervals during the control perfusion and thenat 10-min intervals until the end of experiment. In calculating
the CFR for the 1st 5-min of reperfusion, the volume of thecoronary effluents at 1-min and 4-min were added together
and their sum taken as the volume for the 5-min interval. All
values were calculated as mean ± SD but the SD has been93

omitted to avoid confusion. All parameters were analyzed bymeans of time-series analysis of variance, and then the
differences between the mean value in the control group andthe corresponding values in the other experimental groups ateach measuring time were analyzed using the Dunnett
multiple comparison-test. A p<0.05 was considered as
significant.

Results
Cardiac functions
Coronary flow rate (CFR)
The mean CFR in the control group gradually decreased
from about 17 mL/min to 15 mL/min during the control
perfusion. Just after the onset of reperfusion, CFR started toincrease. Between 5 and 10 min, it recovered to a level nearthat in the control perfusion period, and then showed a slightdecrease until the end of reperfusion. With the change tomKH buffer containing HbV or EV for the last 10 min of thecontrol perfusion in the respective groups, though there wasa larger decrease in the mean CFR in the HbV 0.33 g/dLgroup and EV 0.33 g/dL group than in the control group, thisdifference was not significant. Changes in CFR in thereperfusion period were similar in the control andexperimental groups（Fig. 2.）.

Heart rate (HR)Mean HR values in the control group were maintained atabout 260 beats/min during the control perfusion. HR was
not affected by perfusing with mKH buffer solution
containing HbV or EV for the last 10 min of the control
perfusion. During the reperfusion period, there was no
beating in any hearts of the control group apart from slightbeating in one heart at 10 min. While the mean HR in the EV
0.10 g/dL group was similar to that in the control group,
there was a slight recovery in HR in the EV 0.33 g/dL group,though this difference was not significant as compared to themean HR in the control group. However, there was asignificant recovery in HR in the two HbV groups（HbV 0.10
and 0.33 g/dL）（p<0.05）compared with the control group at
all measurement times during reperfusion. The mean HR in
the HbV 0.33 g/dL group had recovered to the control
perfusion level after 30 min of reperfusion（Fig. 3.）.

Fig. 3. Change in heart rate（HR）during experimental period○: control group, □: HbV 0.10 g/dL group, ■: HbV 0.33 g/dLgroup, △: EV 0.10 g/dL group, ▲: EV 0.33 g/dL group*p<0.05, vs. control group by Dunnett multiple comparison test

Fig. 2. Change in coronary flow rate（CFR）during experimental period○: control group, □: HbV 0.10 g/dL group, ■: HbV 0.33 g/dLgroup, △: EV 0.10 g/dL group, ▲: EV 0.33 g/dL group

94

Left ventricular endodiastolic pressure (LVEDP)
During the control perfusion, the mean LVEDP in thecontrol group was maintained at about 5 mmHg. This value
was not altered by perfusing with the mKH buffer solutions
containing HbV or EV for the last 10 min of the control
perfusion period. After the onset of ischemia, the mean
LVEDP in the control group started to rise gradually, and at
30 min of ischemia it had reached about 40 mmHg. Thechanges in mean LVEDP in the two EV groups were similar

人工血液 Vol. 14 , No.3, 2006

to that in the control group during ischemia. The mean
LVEDPs in the 2 HbV groups seemed to have risen to alesser extent than that in the control group at 30 min ofischemia, though this difference was not significant whencompared with the control group. During reperfusion, themean LVEDPs rose further to 80-90 mmHg in the control and
EV 0.10 g/dL groups, and this rise was maintained until theend of reperfusion. In the EV 0.33 g/dL group, the LVEDP
had risen to about 80 mmHg at 10 min of reperfusion andthen decreased to about 65 mmHg at the end of reperfusion,
though the latter LVEDP was not significant when comparedwith the corresponding value in the control group. In the 2
HbV groups, rises in the mean LVEDP（30-60 mmHg）were
significantly（p<0.05）suppressed as compared with thecontrol group（70-100 mmHg）at 20 and 30 min of reperfusion（Fig. 4.）.

all measurement times. While there appeared to be arecovery in LVDP in the EV 0.33 g/dL group, this was not
significant when compared with the control group. In
contrast, at 20 and 30 min of reperfusion, there was asignificant（p<0.05）recovery in the mean LVDP in both HbVgroups as compared with the control group（Fig. 5.）.

Fig. 5. Change in left ventricular developed pressure（LVDP）duringexperimental period○: control group, □: HbV 0.10 g/dL group, ■: HbV 0.33 g/dLgroup, △: EV 0.10 g/dL group, ▲: EV 0.33 g/dL group*p<0.05, vs. control group by Dunnett multiple comparison test

Fig. 4. Change in left ventricular endodiastolic pressure（LVEDP）duringexperimental period○: control group, □: HbV 0.10 g/dL group, ■: HbV 0.33 g/dLgroup, △: EV 0.10 g/dL group, ▲: EV 0.33 g/dL group*p<0.05, vs. control group by Dunnett multiple comparison test

Left ventricular developed pressure (LVDP)
During the control perfusion, the mean LVDP in thecontrol group gradually decreased from about 175 mmHg to
155 mmHg, and a similar decrease was observed in the otherexperimental groups. During reperfusion, apart from thedevelopment of a small amount of pressure in 1 heart at 10
min, no recovery in LVDP was observed in any heart in thecontrol group, and the mean LVDPs in the EV 0.10 g/dLgroup were nearly the same as those in the control group at

ARTIFICIAL BLOOD Vol. 14 , No.3, 2006

Lactate concentration of coronary effluent
During the control perfusion, there was a gradual increasein the mean lactate concentration of the coronary effluent in
the control group, from about 10 μg/mL to 18 μg/mL.
Though the increase in mean lactate concentration seemed tobe slightly suppressed for perfusion with mKH buffer
containing EV 0.10 g/dL or 0.33 g/dL, the difference from thecontrol group was not significant. In the HbV 0.10 g/dL and
0.33 g/dL groups, the mean lactate concentrations for 5 minafter the onset of HbV perfusion were significantly（p<0.05）
lower than the corresponding control value（Fig. 6.A）.
During reperfusion, there was a sharp increase in thelactate concentration of the coronary effluent in the control
group in the first minute. In the next 4 min of reperfusion, it
decreased rapidly to around the level in the control perfusion,and then continued at about this level until the end ofreperfusion. Changes in the mean lactate concentration in the
HbV 0.10 g/dL group were similar to those in the control
group throughout the reperfusion period. The mean lactate95

concentration in the HbV 0.33 g/dL group at 1 min ofreperfusion was significantly（p<0.05）lower than that in thecontrol group（ Fig. 6.B）. Though the mean lactate
concentration in the HbV 0.33 g/dL group seemed to beslightly higher than that in the control group at 30 min ofreperfusion, the difference was not significant.

Fig. 6. Change in lactate concentration of coronary effluent duringexperimental period
A: Data obtained during control perfusion, B: Data obtained
throughout the experiment. ○: control group, □: HbV 0.10 g/dLgroup, ■: HbV 0.33 g/dL group, △: EV 0.10 g/dL group, ▲: EV 0.33
g/dL group*p<0.05, vs. control group by Dunnett multiple comparison test

96

Discussion
When an organ or a tissue is perfused with a buffer
containing microcapsules like HbV and EV, we must beaware of the possibility of such microcapsules causing
embolisms in the organ or tissue. Nakai et al.10）reported thatwhen isolated rat hearts were perfused with Krebs-Henseleit（KH）buffer containing hemoglobin-encapsulated liposomes（neo red cells, NRC）
, there was a sudden increase inperfusion pressure just after perfusion began, and their
histological findings showed that embolisms were the likely
cause of the increase in perfusion pressure. Further
investigation revealed that inorganic crystals were formed
after mixing NRC with the buffer, and these researchers
suggested that the crystals were the cause of the embolisms.
In this connection, Sakai et al.11）showed that HbV caused noconstriction of resistance arteries or hypertension in a
conscious hamster model.
In the present study, we carefully observed cardiac
functions while the hearts were perfused with mKH buffer
containing HbV or EV for 10 min prior to ischemia. As
mentioned in the results section, cardiac functions（CFR, HR,
LVEDP, and LVDP）were not significantly affected by
perfusing with mKH buffer containing HbV 0.33 g/dL or EV
0.33 g/dL. These results suggest that in contrast to the above
research using NRC, embolisms did not occur in our study.
Our findings with the higher concentrations of HbV and EV,
however, require further clarification.
It is interesting that the lactate concentration of thecoronary effluent was lower in the HbV 0.33 g/mL groupthan in the control group during the first 5 min of HbV
perfusion and first minute of reperfusion. Pyruvate, the final
substrate in the glycolytic pathway, is mainly oxidized in the
mitochondria and partially converted to lactate by lactic
dehydrogenase during control perfusion. Then, in ischemia,
almost all pyruvate is converted to lactate since
mitochondrial oxidation immediately stops, and this lactate isthought to be a factor in cardiac cell injury and delay in therecovery of cardiac functions during reperfusion. Therefore,
we surmise that HbV suppressed lactate production in the
cardiac cells during HbV perfusion and ischemia bystimulating mitochondrial oxidation, resulting in thesignificant recovery in cardiac functions in the HbV 0.33 g/dLgroup during reperfusion, though the mechanism by which
HbV suppresses lactate production remains to be clarified.
In conclusion, our results suggest that HbV altered cardiac
metabolism both before and during ischemia, and as a result,enhanced recovery of cardiac function during reperfusion.

人工血液 Vol. 14 , No.3, 2006

Acknowledgements
This study was supported by Health Sciences Research
Grants（Research on Pharmaceutical and Medical Safety,
Artificial Blood Project）; Ministry of Health, Labor, andWelfare, Japan. Hemoglobin-vesicle and empty-vesicle werekindly supplied by Oxygenix Co., Ltd. A part of this study
was reported at the 13th annual meeting of the Society of
Blood Substitutes, Japan on August 25, 2006.

References
１. Chang TMS. New generations of red blood cell
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２. Squires JE. Artificial blood. Science 2002;8:295:1002-1005.
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４. Takeoka S, Ohgushi T, Sakai H, Nishide H, Tsuchida E.
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５. Izumi Y, Sakai H, Hamada K, Takeoka S, Yamahata T,
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ARTIFICIAL BLOOD Vol. 14 , No.3, 2006

Tsuchida E. Systemic and microvascular responses to thehemorrhagic shock and resuscination with Hb-vesicles.
Am J Physiol Heart Circ Physiol 2002;283:H1191-H1199.
７. Sakai H, Horinouchi H, Tomiyama K, Ideka E, Takeoka S,
Kobayashi K, Tsuchida E. Hemoglobin-vesicles as oxygen
carriers: Influence on phagocytic activity andhistopathological changes in reticuloendothelial system.
Am J Pathol 2001;159:1079-1088.
８. Sakai H, Hamada K, Takeoka S, Nishide H, Tsuchida E.
Physical properties of hemoglobin vesicle as red cell
substitutes. Biotechnol Progr 1996;12:119-125.
９. Lowry OH, Passonneau JV. In: A flexible system of
enzymatic analysis. New York: Academic Press, 1972;194196.
10. Nakai K, Usuba A, Ohta T, Kuwabara M, Nakazato Y,
Motoki R, Takahashi TA. Coronary vascular bed perfusion
with a polyethylene glycol-modified hemoglobinencapsulated liposome, neo red cell, in rats. Artif. Organs
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11. Sakai H, Hara H, Yuasa M, Tsai AG, Takeoka S, Tsuchida
E, Intaglietta M. Molecular dimensions of Hb-based O2
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97●編集後記●私が編集委員になってから2年が経過した．武岡編集長はじめ編集委員各位，研恒社の担当者が本誌内容の充実のため献身的に作業されていることを目の当たりにした．私も微力乍らお役に立てるよう努力していきたい．さて，海外研究者に投稿を依頼することになり，私の留学先であったカリフォルニア大学
サンディエゴ校（UCSD）の研究者二名に御願いし，本号の掲載にこぎつけた．Dr. Hangai（半谷先生）の総説は，第11回
日本血液代替物学会年次大会（札幌）のシンポジウム講演の内
容 に 基 づ い て い る ． Dr. Hangaiは 昨 春 U C S D を 離 れ た が ，

98

PEG-albuminの新展開の礎を築き，UCSDでのmilestoneとなった．米国での今後のご活躍を期待したい．また，微小循環系の血流制御と酸素輸送に関する生理学の権威Prof. Johnsonが第12回大会（東京）で教育講演をされたことを受け，総説の執筆を御願いしたところ御快諾頂いた．Prof. Johnsonは昨夏UCSDを退官されたが，マイペースで研究を継続しておられるとのことである．永年に亘るProf. Johnsonの御業績に敬意を表したい．
編集委員：酒井宏水

人工血液 Vol. 14 , No.3, 2006

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論文，関連する情報，学会会員のための会
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１. 太田和夫. 移植医療と社会. 医学のあゆみ 1993;164:442-6.
２. 砂本順三, 岩本 清. リポソームの調製. 野島庄七, 砂本順三, 井上圭三 編.
リポソーム. 東京: 南江堂,1988;21-40.
３. Fowler SA, Andracki M, Hurst G,
Honkan VA, Walder J, Casteel DA.
Prolongation of the intravascularretention of hemoglobin modified
with a long-chain fatty acidderivative. Artif Cells Blood Substit
Immobil Biotechnol 1994;22:27-42.
４. Reichert CM, Kelly VL, Macher
AM. Pathologic features of AIDS.
In: DeVita VT Jr, Hellman
S,Rosenberg SA, eds. AIDS.
Philadelphia: Lippincott, 1985;111-60.
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