627
Ind. Eng. Chem. Fundam. 1986, 25, 627-633
Literature Cited Achenie, L. E. K.; Biegler, L. T. EDRC Report, 1986; Carnegie-Mellon University, Pittsburgh, PA. Ascher, U.; Christiansen, J.; Russell, R. D. Mafh. Compuf. 1979, 33, 659-679. Bielger. L. T.; Cuthrell, J. E. Comput. Chem. Eng. 1985, 9 ,257-267. Bryson, A. E.: Ho, Y.-C. Applied Optimal Control; Hemisphere: New york, 1975. Carberry. J. J. Chemical and Catalytic Reaction Engineering; McGraw-Hill: New York, 1976. Chitra, S.P.; Govind, R . Chem. Eng. Sci. 1981, 36, 1219-1225. Chitra, S.P.: Govind, R . AIChE J. 1985, 3 1 . 177-193. Conti, G. A. P.; Paterson, W. R . Inst. Chem. Eng. symp. Ser. $985, ,v0,
Jackson, R . J. O D ~Theor. . A m / . 1968, 2. 240-259 Neyfeh, A. Pertuibation MethGs; Wiley: New York, 1973. Nishida, N.; Stephanopoulos, G.: Westerberg, A. W. AIChE J. 1981, 27, 321-350. O'Malley, .~R. E. Introduction to Singular Perturbations; Academic: New York, 1974. PaYnter, J. D.; Haskins, D. E. Chem. Eng. Sci. 1970, 25, 1415-1422, Powell, M. J. D. Lect. Notes Math. 1977, No. 630, 144-155. Ravimohan, A. L. J . Opt. Theor. 1971, 8 , 204-211. Sargent, R. W. H.; Sullivan, G. R. Proc. 8th I F I P Conf. O p t . Tech. 1977, Part 2, 158-168. Trambouze, P. J.; Piret, E. L. AIChE J. 1959, 5 , 384-390. Van de Vusse, J. G. Chem. Eng. Sci. 1964, 79,994-999.
92 . - , 391-402 -.
Danckwerts. P . V . Chem. Eng. Sci. 1953, 2, 1-18. Han, S.-P. J. Opt. Theor. Appl. 1977, 22, 297-309. Horn, F. J. M.; Tsai, M. J. J . Opt. Theo. Appl. 1967, 7 , 131-145.
Received for review June 18, 1986 Accepted July 18, 1986
Changes in Shear-Induced Hemolysis due to Blood Additives: Synthetic Biopolymers and Asthma Drugs Robert C. Lljana,+Joseph M. Monroe,$and Mlchael C. Williams" Chemical Engineering Department, University o f California, Berkeley, Berkeley, California 94720
Mechanical fragility of human red blood cells was evaluated by shearing the blood between rotating polyethylene disks and measuring the timedependent release of hemoglobin. Several blood additives were tested for their effect on this hemolysis: (a) two synthetic polynucleotides (polyadenylate and polycytidylate) as biopolymers with purine and pyrimidine moieties, respectively; (b) two low-molecular-weight purine derivatives (the drug theophylline and uric acid). It was found that polyadenylate always increased hemolysis, polycytidylate often reduced it, theophylline always reduced it, and uric acid was always ineffective. Drug localization data on theophylline showed a large uptake of the additive by cell membranes, the degree of hemolysis protection being proportional to the mass of the drug absorbed. Supplementary cell characterization by resistive pulse spectroscopy documented that the protective drugs caused cell volumes to increase, deformability to decrease, and osmotic fragility to decrease. Chemical and mechanical mechanisms for changes in mechanical fragility are proposed.
Introduction Flow of blood through artificial organs and implanted conduits leads to various forms of blood degradation. The release of erythrocyte hemoglobin is often used as a general measure of red cell damage. In previous work, this laboratory has examined how flow-induced hemolysis is influenced by hydrodynamic factors (Shapiro and Williams, 1970) and by the roughness (Monroe et al., 1981) and surface composition of conduit materials (Offeman and Williams 1979a; Monroe et al., 1980: Offeman and Williams, 1976). Reviews of the topic have also appeared (Bernstein, 1971; Blackshear, 1972; Hellums and Brown, 1977). Few investigations have been directed toward the chemical features of blood that dispose it to hemolyze in shear flow. Hemolysis tendencies have been correlated with preshear plasma chemistry (Offeman and Williams, 1976), statistical correlations being found with elevated concentrations of lactate dehydrogenase (LDH), very low density lipoproteins, and uric acid. A subsequent study (Monroe et al., 1980) of plasma chemistry changes during in vitro shear-induced hemolysis revealed a drop in glucose and rise in uric acid (both presumably due to shear-induced metabolic activity), while Ca2*and Na+ entered the cells and LDH was released. Procter and Gamble Co., Miami Valley Laboratories, Cincinnati, OH. *Union Oil Company, Los Angeles, CA.
The concept that chemical additives to the plasma might reduce hemolysis by some sort of protective action on erythrocytes is relatively new. Blood preservatives have been designed to preserve essential features of cell viability but not with attention to the tendency to hemolyze in subsequent shear flow. The introduction of adenosine into some blood preservatives did, however, reduce such hemolysis (Tadano et al., 1977). It seems likely that other additives could have similar or superior results, and this possibility was the principal motivation for our extended work on the effects of blood additives on shear-induced hemolysis. Studies of plasma proteins, used for precoating synthetic surfaces and added to red cell suspensions, indicated that these substances also afforded hemolytic protection (Nichols and Williams, 1976). Their role in the precoating tests was clear: they transformed the synthetics into surfaces resembling biological ones which induced less chemical trauma in red cells contacting them. The beneficial result of adding proteins to the fluid environment of erythrocytes was less clear. (The most successful was y-globulin.) In a medium deprived of proteins, enrichment by protein supplementation is a step toward restoring a normal (physiological) environment and, thus, returning the cell condition to one of normal hemolytic resistance. Still, this picture fails to address the specifics of chemical interaction between protein and cell that lead to hemolytic resistance. The present study, therefore, was also designed to investigate the role of simple homopolymers, synthesized from a single bio-"mer", having far fewer chemical com-
0196-4313/86/1025-0627$01.50/00 1986 American Chemical Society
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plications than real proteins. Thus, we describe results of testing the following substances: (a) Synthetic polynucleotides, polyadenylate (Poly-A), and polycytidylate (Poly-C). Poly-A carries a purine (adenine) moiety in its nucleotide repeat unit and, surprisingly, proves always to increase hemolysis. Poly-C has a pyrimidine (cytosine) moiety and is never harmful, often reducing hemolysis substantially. (b) Theophylline. Theophylline, a drug commonly used to treat asthmatic conditions, is a purine derivative (xanthine character). Unlike the polypurine (Poly-A),it always reduces hemolysis. (c) Uric acid, added above physiological levels. It is included because it is also a purine derivative, but not methylated like theophylline. The earlier report (Offeman and Williams, 1976) of its being statistically correlated with enhanced tendencies toward hemolysis contributed to its choice in this study. Elsewhere we describe the effects of low-molecular-weight antibiotics on shear-induced hemolysis (Lijana and Williams, 1986). To link results and yield a comparative basis, the present work contains also a direct detailed comparison between two dissimilar drugs: the antibiotic amikacin and the nonantibiotic theophylline.
Experimental Section Blood. Fasted adult males donated blood at Alta Bates Hospital in Berkeley. This blood, preserved with citrate-phosphate-dextrose (CPD), was then kept in our laboratory at 4 OC until testing. Additional samples were sometimes obtained from the local blood bank after legal expiration. All samples were screened to verify normalcy. Additives. Theophylline solution (aminophylline injection) was obtained from Elkins-Sinn Inc. Crystalline powders of reagent-grade uric acid, polyadenylate, and polycytidylate from Miles Laboratories were later dissolved in saline for addition to blood. For surface-coating experiments, solutions of the polymers were prepared in distilled water. Chemical structures of all the additives used in this study are given in Figure 1. Apparatus. Blood was sheared for 5-000 s between the smooth polyethylene (PE) surfaces of a rotating-disk device. Flow was laminar, with the maximum shear stress (130 dyn/cm2, at the rim) being well below the threshold ( ~ 1 5 0 0dyn/cm2) for cell rupture (Nevaril et al., 1968). Geometrical and operational details are described elsewhere (Nichols and Williams, 1976). Procedure. On the day before shear testing, the blood was gently filtered and collected as 30-mL samples in polypropylene tubes. In most tests, the dissolved additive was introduced a t this time in solution form and allowed to incubate overnight a t 4 OC. In another set of tests, the additive was injected after the blood had been loaded into the shearing device, just prior to flow. Saline concentrations of the additives to be incubated were adjusted so that the same small volume of the solution (about 0.5 mL) could be added to the blood to provide any desired plasma concentration, c. For tests involving the coating of PE disks, coating solutions of the polynucleotides were prepared in distilled water with the same molar concentration of single-phosphorus repeat groups, 6.25 pmol of P / m L (meaning a total mass concentration of 2.06 mg/mL for Poly-A and 1.91 mg/mL for Poly-C). Identical concentrations were prepared in saline for injection runs. Coating of the P E disks with Poly-A and Poly-C was effected by placing the disks face-down for 24 h in Petri dishes containing the solutions. Polymer adsorption on
Y 2
I
H2N-CHZ
I
H$OH
Figure 1. Chemical structures of additives. (a) Polyadenylate (Poly-A). (b) Polycytidylate (Poly-C). (c) Theophylline. (d) Uric acid. (e) Amikacin.
the disk face and rim was verified by staining. Prior to shearing, blood samples were warmed to room temperature over about 30 min and then loaded into the room-temperature disk device as described elsewhere (Lijana and Williams, 1986). On a typical test day, as many as five shearing runs could be made with samples of the same blood having different additives and/or concentrations. Samples taken at five times during a run were centrifuged immediately, and the plasma was segregated for later chemical analysis. Kinetic Hemolysis Curve (KHC). The KHC is a graph representing the plasma hemoglobin concentration, C, as a function of the duration of shear, t. It is convenient to work with the incremental change, AC(t) = C ( t )- C(O), and then normalize AC to adjust for minor variations of hematocrit H and blood volume V in the device from run to run
where Hsd = 0.35 and V = 22.0 mL. For comparisons between several KHC, it is useful to characterize each in terms of its slope, u, at the beginning ( t = 0) and end ( t = tf) of the run: go I
(dAC,,d/dt),=,
of E (dAC,,,/dt),=,,
(2)
Because the KHC varies with blood age and donor, it is also necessary to make a final normalization with reference values of u taken from control runs (i.e., without additives) on the same blood: This permits results with bloods of different ages and from different donors to be compared in terms of the ranking parameters c and E.
* 4
I601
1201
“std (mg/dl)
40
OO
Ind. Eng. Chem. Fundam., Vol. 25, No. 4, 1986 629 I 00
1
B L O O D : 8x6146 SHTLOMRAATG0EC R A ;GTE: ’ 24 41%days /ioa+ed
80
60 40
0v o ’ 0
Time (sec)
Figure 2. Kinetic hemolysis curves. Blood identity, age a t time of shearing, and hematocrit are given. Additives are also specified.
POLY-A
BLOOD STORAGE AGE HEMATOCRIT
20
6000
4000
167. p g l m l -
1 6 . 7 pg/ml
C O N T R O L ( 0 ) 8. P O L Y - C C o a t e d (0)
2000
’ CONTROL
I THEOPHYLLINE
I 2000
c xX33 77 7 3 C 2 4 doys 4 2 .5 %
I
I
I
.
-
4000
6000
T i m e (secj
Figure 4. Kinetic hemolysis curves. Blood identity, age a t time of shearing, and hematocrit are given. Additive and its initial plasma concentrations are also specified.
Injected
’
I
/
’
I
PURINE DERIVATIVES
I
I POLYADENYLATE
6oF 80
40. “std (mg/dl)
’
Physiological uric acid: 46.7 p g h l
20 ’ BLOOD: W X 6 7 1 0 S T O R A G E AGE: 16 days H E M ATOCR I T : 4 I %
I
2000
I
I
4000 Time (sec)
BLOOD‘ C X 4 0 9 4 STORAGE AGE 23 days HEMATOCRIT 41 0 % I
I
I
6000
Figure 3. Kinetic hemolysis curves. Blood identity, age a t time of shearing, and hematocrit are given. Additives are also specified.
Erythrocyte Characterization by RPS. Resistive pulse spectroscopy (RPS) was used to obtain red cell size distributions, deformability, and dynamic osmotic fragility (Me1 and Yee, 1975; Yee and Mel, 1978). Data were obtained before and after shearing, on bloods with and without additives. We summarized and illustrated the method very recently (Lijana and Williams, 1986); briefly, the cell size distribution has a peak identified as the modal cell volume, Vc (in units of RPS channel numbers), and “deformability curves” (volume distribution curves distorted by influence of hydrodynamic stress on deformable particles) are characterized by a uniquely defined dimensionless ratio-the deformability index, DI-that takes on values slightly greater than 1.0 for deformable particles and moves toward zero with increasing particle rigidity. Osmotic fragility reflects cell resistance to the isotropic stress imposed by osmotic imbalance across the cell membrane; it is a continuous time-dependent RPS test (several minutes of cell exposure to hypotonic saline) yielding the change in percentage of cells that are ghosts (the “ghost percentage”, GP) as cell lysis and repair of the ghosts proceed. Results Direct comparisons of Poly-A and Poly-C effects on the KHC are seen in Figure 2 (disk adsorption of polymers) and Figure 3 (polymers injected into blood before test). For tests using blood incubated with the various additives, the KHC’s for representative runs are shown as follows: theophylline (Figure 4) at several concentrations; contrasts between the purine derivatives Poly-A, theophylline, and uric acid (Figure 5) a t the same molar concentration of purine moieties; contrast between the drugs theophylline and amikacin (Figure 6) a t the same molar concentration. The ranking parameters, e and E, for the incubated bloods in all tests are given in Table I. Measurements with RPS were made primarily in conjunction with the theophylline/amikacin comparison,
0
I 4000
I
2000
Time
I
I 6000
I
(sec)
Figure 5. Kinetic hemolysis curves. Blood identity, age a t time of shearing, and hematocrit are given. Additives and their initial plasma concentrations are also specified. 1
I
I
I
I
i
I
70 THEOPHY L L I NE -
50 -
-
3 4 I pglrnl AM I K ACI N
“std (rngldl)
BLOOD STORAGE AGE HEMATOCRIT
1
0
4000
2000
-
CX4024 2 3 doys 38.5%
I
.
6000
r i m e (sec)
Figure 6. Kinetic hemolysis curves. Blood identity, age a t time of shearing, and hematocrit are given. Additives and their initial plasma concentrations are also specified. Table I. Purine Ranking Parameters
blood (I.D. no.) CX4094 ABO12-B
age, days 23 11
initial plasma concn, kg/mL 31.7 32.0
uric acid
YX7697 CX4094 YX7697 YX7697 AB013-B AB012-B AB013-B
23 23 23 23 10 11 10
20.1 31.7 70.1 350 20.1 32.0 355
1.00 1.00 1.00 1.00 1.00 1.00 1.00
1.00 1.00 1.00 1.00 1.00 1.00 1.00
theophylline
AB013-B AS3304 CX4024 ABO13-B CX4094 AB013-B ABO12-B
24 23 23 24 23 24
8.3 10.0 13.7 16.7 31.7 167 32.0
0.88 0.84 0.83 0.82 0.79 0.88 0.73
0.87 0.86 0.85 0.85 0.84 0.87 0.71
additive polyadenylate
11
ranking parameters e E 1.19 1.32 2.14 1.48
630
Ind. Eng. Chem. Fundam., Vol. 25, No. 4, 1986
Table 11. Erythrocyte Characterization Parameters blood age, days drug (concentration) (I.D. ___no.)
cx402-1
control theophylline (13.7 pg/mL) amikacin (34.1 Gg/mL)
?3
Deformation index.
modal size unsheared sheared 35.0 34.0 35.8 35.2 37.0 36.8
DI" unsheared 0.98 0.83 0.69
RelatiLe osmotic fragility -
BLGOD STORAGE AGE
yb
sheared 0.86 0.77 0.58
1
I
unsheared 1.00 1.00 1.06
I
I
sheared 0.98 0.88 0.95
I
-Unsheored Sheared I
CX4024 23days
4 unsheared AMIKPCIN I
1
I
1
unsheared CONTROL 8 THEOPHYLLINE
GF
I 8 days 135 mOsm
c $.-.--3'2
i
THEOPWYLLINE
n
65,
c
AMI K A C I V
n sheared
, , GP 7 ,
50
61
P I .
83
IC0
,, /
1sec
Figure 7. Dynamic osmotic hemolysis (GP = ghost percentage) for blood with specified additives and shearing histories.
though some Poly-C systems were tested as well. Spectra on cell size distributions resemble those presented in the full antibiotic study (Lijana and Williams, 1986) and thus will not be given here. Dynamic osmotic hemolysis curves are shown in Figures 7 (drugs) and 8 (Poly-C), demonstrating that G P approaches an asymptote (GP,) that will be taken here as a measure of cell fragility. The derived parameters from RPS-VL, DI, and y 5 (GP,)/ (GP,),,,,,,,I-are listed in Table 11.
Discussion Polynucleotides. The performances of the synthetic homopolymers Poly-A and Poly-C were very different and not simply related to the behavior of biological proteins in shearing tests. In all the disk-coating experiments (e.g., Figure 2), Poly-A coatings increased hemolysis by large amounts-often by more than 10G% relative to initially clean P E disks-and Poly-C coatings behaved essentially like the PE substrate. There was no assurance that these performances reflected purely surface-bound polymer actions; it seemed possible that some of the adsorbed polymer was washing off during flow and exerting its function while in solution. This prompted the tests with directly injected polymer solutions. The same amounts of polymer were exposed to the blood in both types of experiments (3.1pmol of P / 2 2 mL of blood) if one assumes that the precoated disks adsorbed all the polymer from solution. Again Poly-A was always hemolytic, but now Poly-C demonstrated different behavior. Figure 3 shows that Poly-C could sometimes provide substantial protection against hemolysis, while other runs showed it to be neutral. The collection of these data, together with RPS evidence, suggested strongly that a range of blood response was seen with Poly-C because of highly donor-specific variables. In no case, however, was Poly-C found to be associated with increased mechanical hemolysis in shear flow, and in no case was Poly-A less than damaging. Incubation of blood with Poly-A reinforced this picture (Figure 5 ) . The hemolytic behavior of Poly-A is unlikely to be related in a simple way to its purine moiety, since the other purine derivatives tested here (see below) did not behave in this fashion. Its action may stem from its polymeric nature, paralleling that of the antibiotic polymer amphotericin B, which is similarly linked to an amino sugar and known to damage erythrocyte membranes by forming
9 days I 3 4 mOsm
0
40
80 T (seci
Figure 8. Dynamic osmotic hemolysis (GP = ghost percentage) for blood with specified additive and shearing histories.
pores in the cell's lipid bilayer (Rubin et al., 1972). A similar mechanism may occur with the polypeptide antibiotic gramicidin c. It is believed (McLaughlin and Eisenberg, 1975) that these molecules dimerize to form a hollow helix with a hydrophobic exterior that allows them to penetrate the lipid bilayer to form pores with a hydrophilic interior for escape of cell contents. Cells leak first K+ and then hemoglobin (Butler and Cottlove, 1971), in proportion to the drug concentration. The very different behavior of Poly-C can perhaps be related to its pyrimidine moiety, making it incapable sterically or energetically of generating a membrane tunnel (if, indeed, this is what Poly-A does). I t is also possible that the carboxylic oxygen on its pyrimidine ring makes it too hydrophilic to pierce the lipid membrane or perhaps too tightly hydrogen bonded internally to extend into a helix configuration to create a pore. Why Poly-C should offer protection a t all is not immediately evident, but it was shown (Lijana and Williams, 1986) that protective antibiotics moved strongly to erythrocyte membranes. It seems possible that Poly-C does likewise, adsorbing at external hydrophilic sites and forming a mechanical layer with strengthening characteristics. Small-Molecule Purine Derivatives. Total ineffectiveness of the trioxypurine, uric acid, was seen at all concentrations (e.g., Figure 5). Table I shows that plasma levels as high as 350 pg/mL above physiological levels had no effect whatever on the hemolytic properties of either old or fresher blood. This agrees with a second correlative study (Offeman and Williams, 1979b), using more extensive data than the earlier one (Offeman and Williams, 1976) that reported an association of hemolytic propensity with physiological uric acid levels. The latter study was probably based upon insufficient sampling and possibly
Ind. Eng. Chem. Fundam., Vol. 25, No. 4, 1986 631 Table 111. Drug Localization Studies ulasma concentrations of drug change, preshear blood (I.D. no.) AS3304O CX4024" AS3304 CX4024
drug theophylline amikacin
age, days 23 23 23 23
added, pg/mL 10.0 13.7 16.8 34.1
pg/mLb -2.7 -3.3 -2.6 -5.5
% -27.0 -24.0 -15.5 -16.1
change, postshear pg/mLb -2.1 -2.5 -2.6 -3.6
70 -21.0 -18.2 -15.5 -10.6
pg/mLC +0.6 +0.8 0 +1.9
% +6.0 +5.8 0 +5.6
"Hemolytic ranking parameters from the KHC are listed in 7'able I. *Change relative to the original concentration, added prior to incubation. Change relative to preshear incubated levels. i .oo
I
1
I
I I I I , ,
1
I
I 1 l l l l
BL!OODS:
0,951
Amikacin\
e~ ~ 3 3 0 4
'\\Gentamicin
\
\
1 1 1 1 1 1 1
Q AB0 I 3 8
0 CX4094
\
\Streptomycin \
,
\
\
i
\
\
'.
\
\ I
0.75 I
1
I
l l l l l l
10
I
I
I
\
\
I l l l l l l
'
\
I
l \ l I I I I I
100
1000
c , pg a d d i t i v e / m l plasma ( i n i t i a l )
Figure 9. Dependence of the shearing hemolysis parameter E (see text) on drug Concentration. Data points and solid line represent theophylline, for bloods stored 23-24 days. Dashed curves give antibiotic performances with bloods stored 23-27 days for comparison (Lijana and Williams, 1986).
related to anomalous donor conditions. That uric acid does not enhance hemolysis is not surprising, in retrospect, in view of its normal physiological presence and the fact that patients with gout have normal plasma hemoglobin levels. This also reinforces the idea that the basic purine chemical structure (Figure 1)is not intrinsically harmful to erythrocytes. The methylated dioxypurine, theophylline, proved to be capable of hemolysis reduction; all values of e and E were less than unity (Table I). However, the concentration dependence of this protective effect was not monotonic. As shown in Figure 9, the function E(c) for theophylline has a minimum and a t higher dosages the protection is reduced. This is unlike the performance of the antibiotics (Lijana and Williams, 1986), represented in Figure 9 by dashed lines. Despite this apparent reversal of behavior at high c, theophylline is still quite effective in reducing hemolysis ( E z 0.85). It is not unlike penicillin in this regard, both as to level of performance and low concentration threshold of effectiveness. Comparisons among the three purine derivatives can be evaluated from Figure 5 in a direct chemical sense because (a) all were added in the same set of experiments and (b) the amounts added increased the plasma purine-group concentration by the same amount (32 pg/mL). While Figure 5 applies to old blood, the same relative performance among the three additives was found also for fresher blood. However, some dependence on blood storage age was seen: all tendencies, whether protective or damaging, were amplified in the E and t values for fresher blood. Table I shows that the hemolytic Poly-A was relatively more damaging for fresher blood, while the hemolysis-suppressing theophylline was relatively more protective for fresher blood. The latter is well illustrated at c r 32 pg/mL, which produced E = 0.71 in 11-day-old blood but only E = 0.84 in 23-day-old blood. This enhanced relative
protection with theophylline in fresh blood is opposite to our findings (Lijana and Williams, 1986) with the antibiotics (penicillin, amikacin). Indeed, this particular performance of theophylline with 11-day blood ( e = 0.73, E = 0.71) was the best seen in any of the antibiotic and xanthine drug hemolysis tests (short of physiologically toxic concentrations of some of the antibiotics). Drug Localization. Interpretation of results depends on knowing how the additives are distributed in the blood, before and after shearing. This was investigated for theophylline and one antibiotic, amikacin. Two bloods were used; the KHC's for one are shown in Figure 6. Prior to shearing and after shearing, blood samples were taken and centrifuged and the plasma and cellular fractions separated. Plasma and cytoplasm were analyzed for theophylline by either ultraviolet spectrophotometry or high-pressure liquid chromatography and for amikacin by radioimmunoassay. Results are displayed in Table 111. Neither drug was found to be present in cytoplasm, thus requiring that the original dosage was distributed between the plasma and membranous material. (Other tests showed that the drugs were not decomposing in solution or adsorbing onto container walls.) Since plasma concentrations were always reduced by overnight incubation, it is clear that cell membranes were attracting substantial amounts of the additive. Table I11 shows that the fraction of drug attached to membranes was greater for theophylline ( ~ 2 5 % than ) for amikacin (=16%), for both bloods and for two different levels of dosage. This greater binding effectiveness demonstrated by theophylline may explain why its concentration threshold for reducing hemolysis is lower than that of amikacin (Figure 9). Particularly significant is the fact that the mass of drug in the membrane seems to correlate with hemolytic protection, without distinction as to chemical character. For example, in Figure 6 the amikacin system had lowest hemolysis, with a membrane loading corresponding to the 5.5 pg/mL that the plasma lost. Somewhat less protection was afforded by theophylline in the same blood, where the plasma lost 3.3 pg/mL. The quantitative aspect of the correlation in this case is quite good; the ratio 3.315.5 is very close to the relative magnitudes by which these two agents reduced hemolysis, as seen in Figure 6. Hemolysis protection does not correlate here with the number of molecules in the cell membrane. In the case just cited, theophylline was present at a level of 2.1 X lo6 molecules/cell, while amikacin (the more effective agent) was a t only 0.5 x lo6. Tests with the older blood (AS3304), using other concentrations, produced much closer results for the two additives than in Figure 6, and this corresponds to the membranes having very similar mass loadings (about 2.7 pg/mL lost from plasma for both drugs). These observations are not intended to suggest that chemical differences in the drugs are not significant-such differences must, after all, determine the affinity between cell membrane and additive that is responsible for the loading differences-but only that the primary causative
632 Ind. Eng. Chem. Fundam., Vol. 25, No. 4, 1986
factor for hemolytic protection may be the bulk or volume of the drug molecules within or on the membrane. The trend of hemolytic protection increasing with c is thus probably tied to the corresponding increase of cell membrane loading; Table I11 shows they are directly proportional to each other. This parallels the antibiotic results (Lijana and Williams, 1986). Table I11 also shows that cells in these old bloods tended to lose a small fraction of their drug loading during shear (i.e., plasma levels were increased after shearing), about 6% for both drugs. Evidence from the antibiotic work, however, indicated that cells in fresher blood can increase their membrane loading during shear. This suggests that cells suffer a loss of drug binding effectiveness with cell storage age; no mechanism for this is known. Cell Size Distributions and Deformability. Table I1 reveals, for the same blood whose KHC’s are given in Figure 6, that modal cell volume, Vc,was increased by incubation with both theophylline and amikacin. This is consistent with the membrane absorption of these drugs cited above and is seen in both the preshear and postshear cases. Interestingly, the fractional increase in preshear pc was precisely proportional to the mass concentration of drug, regardless of drug chemistry: AVc = 0.8 unit for theophylline and 2.0 units for amikacin, a ratio of 0.40 that agrees with the ratio of amounts of drug added (13.7/34.1). The effect of shear itself was always to reduce regardless of whether a drug (or which drug) was present. This probably corresponds to the observed shear-induced loss of potassium that would also extract water in preserving osmotic equilibrium. The loss of hemoglobin itself is too small to account for the observed cell shrinkage in shear. The DI for these 23-day-old cells was lower (0.98) than for fresh erythrocytes, due to loss of flexibility accompanying progressive sphering during storage. Addition of either theophylline or amikacin caused further loss of deformability, presumably due to the combined effects of larger (with membrane area unchanging) and a stiffening of the membrane due to penetration by drug molecules. The DI fell further for theophylline than for amikacin, again correlating well with their relative effects (at these loadings) in increasing Vcand being absorbed by the membranes. Osmotic Fragility. Figure 7 shows the GP(t) evolution for both unsheared and sheared blood samples. For unsheared blood, theophylline had no distinguishable effect but amikacin induced a surprising increase of osmotic fragility. It is commonly believed, on intuitive grounds, that there is a direct relationship between osmotic and mechanical fragility; if so, these osmotic results would suggest that bloods incubated with these additives would then give parallel results in shear-induced hemolysis. However, Figure 6 shows this not to be so; the amikacinloaded sample had a lower KHC than the theophyllineloaded one. It is clear that the two experiments measure different types of fragility. For the sheared bloods in Figure 7, theophylline produced a large reduction of osmotic fragility, while even amikacin was effective but significantly less so. Thus, in both the sheared and unsheared cases, the value of GP, for amikacin blood exceeded that for theophylline blood; theophylline was, in this sense, the more protective (or less damaging) agent. Because this is opposite to their order of effectiveness in reducing shear hemolysis (Figure 6), we must conclude that membrane trauma suffered by cells in shear flow differs from trauma suffered under osmotic stress.
v,,
vc
Differences in osmotic responses between cases involving different additives can be explained by the greater cell uptake of amikacin than of theophylline (e.g., for sheared blood (2x4024, 3.6 vs. 2.5 hg/mL of plasma; from Table 111). This leads to larger and less deformable cells with amikacin (Table 11),rendering them less capable of further swelling before critical volume is achieved and osmotic rupture occurs. Similar osmotic fragility tests were made on bloods injected with Poly-C, an agent that had demonstrated variable but often protective effects in shear flow (Figure 3). Figure 8 displays the behavior of Poly-C in tests with a blood for which it also caused reduction in the KHC. With 9-day-old blood, the role of Poly-C was to reduce osmotic fragility for both unsheared and sheared samples; in this sense, its performance was better than either theophylline or amikacin shown in Figure 7. However, changes occurred as the blood aged; when the blood was 18 days old, Poly-C still induced a reduction in GP in the unsheared case but an increase in the sheared case (unlike theophylline and amikacin). This may be a manifestation of sensitivity of the Poly-C binding to changes in cell surface chemistry with age in storage. Mechanisms. The protective effect of theophylline, like that of the antibiotics, is no doubt related to loading of cell membranes with the drug. Related observations in the literature are discussed elsewhere (Lijana and Williams, 1986). The effect may be purely mechanical in many cases, due to bulkiness of the intercalated molecules causing lateral membrane stiffness or, perhaps, due to interactions with spectrin at the membrane endoface. A chemical mechanism may also be involved, although cell metabolism is not likely to be affected here as is probably the case when adenosine is added (Tadano et al., 1977). With theophylline, we might propose an alteration of the spectrin network by enhanced phosphorylation: theophylline inhibits catabolism of cyclic adenosine 3‘,5’monophosphate (CAMP)through inhibition of a critical enzyme. Since cAMP influences a number of metabolic processes and influences the degree of phosphorylation of erythrocyte membrane proteins through its actions on a protein kinase, theophylline then may influence spectrin phosphorylation-and therefore membrane deformability-by maintaining erythrocyte cAMP levels. An explanation along these lines for theophylline is consistent with the total ineffectiveness of uric acid, which has a structure extremely similar to theophylline but lacking the identity needed for a parallel interaction with an enzyme. The purine Poly-A is still further from the desired structure, and the pyrimidine Poly-C probably acts without being a chemical mediator at all.
Acknowledgment We thank Dr. H. C. Mel, Department of Biophysics at the University of California, Berkeley, and his associates Sarah Rabinovici and Jon White for their help with RPS measurements. Additional help was provided by Dr. B. H. Lubin of Children’s Hospital, Oakland, and Dr. W. S. Palmer of Alta Bates Hospital, Berkeley. It was a privilege to be advised also by the late Dr. Henry Borsook, who will be deeply missed. Financial support was provided by NIH Grant R01-HL23274 from the Devices and Technology Branch, Division of Heart and Vascular Diseases, of the National Heart, Lung, and Blood Institute. Registry No. a,24937-83-5; b, 30811-80-4;c, 58-55-9; d, 69-93-2.
Literature Cited Bernstein. E. F. Fed. f r o c . 1971, 30, 1510-1515. Blackshear, P. L. I n Biomechanics; Fung, Y. C., Perrone, N., Anliker, M., Eds.; Prentice-Hall: Englewood Cliffs, NJ, 1972; Chapter 19.
Ind. Eng. Chem. Fundam. 1986, 25,
Butler, W. T.; Cottlove, E. J . Infect. Dis. 1971, 723, 341-350. Hellums, J. D.; Brown, C. H. I n Cardiovascular Flow Dynamics and Measurements; Hwang, N. H. C., Norman, N. A,, Eds.; University Park: Baltimore, MD, 1977;Chapter 20. Lijana, R. C.; Williams, M. C. Cell Biophys. 1986, 8 , 223-242. McLaughlin, S.;Eisenberg, M. Annu. Rev. Biophys. Bioeng. 1975, 4 ,
335-366. Mel, H. C.; Yee, J. P. Blood Cells 1975, 7 , 391-399. Monroe, J. M.; Lijana, R. C.; Williams, M. C. Biomafer., Med. Devices, Artif. Organs 1980, 8 , 103-144. Monroe. J. M.; True, D. E.; Williams, M. C. J . Biomed. Mater. Res. 1981, 75, 923-939. Nevaril, C. G.;Lynch, E. C.; Alfrey, C. P.; Hellums. J. D. J . Lab. Clin. Med. 1968, 77,784-790. Nichols, A. R.; Williams, M. C. Biomafer., Med. Devices, Artifif.Organs 1978,
633
633-635
Offeman, R. D.;Williams, M. C. Biomafer., Med. Devices, Artif. Organs 1978, 4 , 49-79. Offeman, R. D.; Williams, M. C. Biomafer., Med. Devices, Arfif. Organs I979a, 7 , 359-391. Offeman, R. D.; Williams, M. C. Biomater ., Med. Devices, Arfif. Organs 1979b, 7,393-420. Rubin, C. S.; Erlichman, J.; Rosen, 0. M. J . Biol. Chem. 1972, 2 4 7 ,
6135-6 139. ShaDiro, S. I.; Williams, M. C. AIChE J . 1970, 76.575-579. Tadano. K.;Hellums, J. D.; Lynch, E. C.; Peck, E. J.; Alfrey, C. P. Blood Cells 1977, 3 , 163-174. Yee, J. P.; Mel, H. C. Biorheology 1978, 75,321-339.
Received for review J u n e 4, 1986 Accepted July 21, 1986
4 , 21-48.
Rates of Blood Filtration. A Brief Review Fumltake Yoshidat Kyoto University, Kyoto 606, Japan
Filtration of blood has two major categories: (a) ultrafiltration (“hemofiltration”) used in some types of artificial kidneys and (b) microfiltration (membrane “plasmapheresis”) used to separate blood into cells and plasma for therapeutic purposes or in pharmaceutical processing. The filtrate fluxes in both (a) and (b), which vary with the channel dimensions and the wall shear rates, can be correlated by assuming the concentration polarization of proteins in (a) and of blood cells in (b). I n (a) the effective diffusivity of proteins in plasma varies with the shear rate and the hematocrit (volume percentage of red cells). I n (b) the effective diffusivity of cells in plasma varies with the shear rate, the cell size, and the hematocrit. Only studies of chemical engineering interest are reviewed.
General Filtration of blood is increasingly important in medical technology. It has two major categories. One is ultrafiltration of blood, i.e., “hemofiltration” in medical terminology. In the artificial kidney using this principle, the blood of a patient is recirculated by a pump through an extracorporeal system which includes an ultrafilter. Blood cells and macromolecules in plasma, such as proteins, do not pass through the filter membrane. The filtrate containing micromolecular solutes, including urea and other uremic toxins, is continuously discarded. The loss of body fluid is made up by diluting the blood returning to the blood vessel of the patient with a physiological saline solution, either before or after filtration. Because of the higher costs of the equipment and the diluting fluid, such a system is not yet widely used, although it has merit over the conventional artificial kidney of the hemodialyzer type in that toxins larger than urea in molecular size can be removed from blood by convective transport through the membrane at the same rates as urea. Of more recent development is the spontaneous continuous “AV (arteriovenous) hemofilter” for the removal of excessive body fluid of patients suffering from edema and other disorders. In this case, the driving forces for blood flow through the filter and for filtrate flow through the membrane are provided by the pumping action of the human heart. It might be mentioned that the glomerular basement membrane of the human kidney is also an ultrafilter. The other category of blood filtration is microfiltration, or, in medical terminology, membrane “plasmapheresis” Professor e m e r i t u s , C h e m i c a l E n g i n e e r i n g . A d d r e s s correspondence to: 2 Matsugasaki-Yobikaeshicho, K y o t o 606, Japan.
(from the Greek word aphairesis meaning removal). In this case, formed blood elements, i.e., erythrocytes (red blood cells (RBC), ca. 8 pm), leukocytes (while blood cells, ca. 10 pm), and platelets (ca. 3 pm), are filtered out by a microporous membrane. The filtrate is plasma, which contains all the macro- and micromolecular solutes outside the cells. Plasmapheresis has two major applications. In donor plasmapheresis, also performed by centrifugation, blood cells are returned to the blood vessel of the donor, and plasma is used for transfusion or large-scale fractionation of plasma components such as albumin. Intensive research is in progress for therapeutic applications of membrane plasmapheresis, in which plasma is continuously separated from the blood of patients with various serious diseases due to abnormality in blood components, e.g., myasthenia gravis, macroglobulinemia, rheumatoid arthritis, hyperleukocytosis, etc. Plasma containing pathogenic molecules is either replaced by plasma from healthy donors or is further treated to remove pathogens before it is returned to the patient. Various methods of plasma treatment, such as adsorption, affinity chromatography, and cascade filtration, are being developed. In cases in which disorder is with blood cells, removal of diseased cells could be performed after plasma separation. For blood ultrafiltration, various anisotropic membranes of polysulfones, poly(acrylonitri1e)(PAN),cellulose acetate, etc., are available. For membrane plasma separation, various mkroporous membranes, normally with mean pore sizes of 0.2-0.6 pm, made of cellulose acetate, PAN, poly(propylene), poly(methy1 methacrylate), poly(viny1 alcohol), etc., are used. Ozawa et al. (1986) report data on plasma separation by ceramic membranes, which could be used repeatedly after regeneration. As filter modules, the hollow-fiber (capillary) type and the flat-membrane type
0196-4313/86/1025-0633$01.50/00 1986 American Chemical Society
