Di-2-Ethylhexyl Phthalate (DEHP) and Mono-2-Ethylhexyl Phthalate (MEHP) Accumulation in Whole Blood and Red Cell Concentrates C. C. PECK,D. G. ODOM,H. I. FRIEDMAN, P. W. ALBRO,J. R. HASS, J. T. BRADYAND D. A. JESS From the Blood Research Division, Department of Surgery, Letterman Army Institute of Research Presidio of San Francisco, and the National Institute of Environmental Health Sciences, Research Triangle Park. North Carolina
Plasma DEHP concentrations w e n measured weekly la whok bloodand red Ccn concentrates(RCC) during 21 days of storage in standard CPD within PL-130 blood bags. In addition, DEHP and MEHP accumulation pattermwere lnvest@td in blood s t d for 42 days in modiAed CPD with adenine wlthin PL-146 and BB-69 storage containers. Total per-unit plasma DEHP of RCC units was 49 to 71 percea of the total In plasma of whok bbod units (PL.130). From 28 to 42 days, IIRM DEHP levels were 12 to 19 per cent higher in whole blood stored In PL-146 than in B W . Although MEHP was aot found in m y blood bag plastic, MEHP accumulated in plasma during whok blood storage. MEHP concentrations were 2.8 to 3.8 times higher In plesnu StOFed In BB49 than h PL-146. It is postulatedthat MEHP a r k from hydrolysis of DEHP by pinsma lipase, even in frozen plasma Mmplcs, and th.t tk n te or this renctbn is -ni by blood bag piastk surface characteristks.
INFUSION of the plasticizer DEHP leached from polyvinylchloride (PVC) bags into blood during cold storage has been considered a possible health hazard.3 To assess the po- tential magnitude of human exposure to this compound, a knowledge of the quantity of DEHP present in units of blood or red cell concentrate after given storage periods is desirable. In the United States, blood and blood products are stored in PVC bags marketed by two manufacturers, but DEHP leaching patterns have been reported only Received for publication February 6 , 1978; accepted March 5 , 1978. The opinions of assertions contained herein are the views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or Department of Defense.
137
for blood stored in one manufacturer's bag.s*13Furthermore, little is known concerning DEHP accumulation in blood preserved in adenine-enriched CPD during extended storage (greater than 21 days). This latter problem is of considerable interest since licensure of adenine fortified blood is being considered.z1 In studies on the metabolic fate of DEHP in primates (African Green Monkey and man), MEHP and oxidation products of MEHP were found in plasma and urine following intravenous administrationof plasma containing leached DEHP.16 While the MEHP compounds were derived primarily from in vivo metabolism of DEHP, small concentrations of MEHP were discovered in the infusion plasmas. This latter finding prompted a search for MEHP in the plastic of PVC blood containers currently in use as well as in plasma during storage. The present investigation was designed to provide the following information: 1) DEHP accumulation in plasma of whole blood and red cell concentrates stored in CPD for three weeks at 4 C in one type of blood storage container; 2) DEHP and MEHP accumulation in blood in PVC containers from two different manufacturers kept for six weeks at 4 C but preserved in adenine-fortified CPD, 3) DEHP and MEHP content in the plastic of unused blood bags from the same two manufacturers; and, 4) the source and accumulationpattern of MEHPduring storage.
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PECK ET AL.
Methods Two blood bag manufacturers fabricated three types of plastic storage containers utilized in this study: PL-130, PL-146 (Fenwal Laboratories, Morton Grove, IL), and BB-69 (McGaw Laboratories, Santa Ana. CA). In Experiment I, the accumulation of DEHP in plasma was determined and compared in four whole blood and four red cell concentrate units (average Hct = 71%), stored in standard CPDI8 for 21 days within PL130 blood bags. In Experiment 11, plasma DEHP and MEHP accumulation were measured in blood stored for 42 days in adenine-enriched CPD within PL-146 and BB-69 blood bags. In both Experiments I and 11, each unit was mixed, sampled (10 ml whole blood) aseptically at weekly intervals, centrifuged, and the plasma was frozen in phthalate free glassware for later analysis. After 87 to 120 days of frozen storage, plasma DEHP concentrations were measured in all samples. Following an identical frozen storage period, plasma MEHP concentrations were measured in 21 and 42 day samples from Experiment 11. In order to assess possible MEHP accumulation during frozen storage (-25 C), one 42-day sample (BB-69) was reanalyzed after 360 days of frozen storage. In addition, 35-day samples from the same experiment which had been frozen for one year were analyzed for MEHP concentration. Finally, Experiment I1 was repeated in two PL-146 and two BB-69 bags, and the samples were analyzed for MEHP immediately after removal from the bag on storage days 21 and 42. After termination of each experiment, the contents of blood bags were cultured. Bacterial contamination was noted in only one case: a red cell concentrate unit in Experiment I, the results of which were discarded. In Experiment 111, phthalate identity and content were determined in strips of PL- 130, PL- 146, and BB-69 plastics cut from unused bags. The inner surfaces (side in contact with blood during storage) of identical strips were subjected to electron microscopic examination. Blood Collection and Storage
One unit of blood was drawn from each of 16 healthy adults who had given voluntary informed consent. The units were stored at 4 C within 30 minutes of phlebotomy.12Red cell concentrate units were centrifuged to a hematocrit of =70 per cent. Two anticoagulants were used: 1) standard citrate-phosphatedextrose(CPD)I8and 2) modified CPD (per 63 ml: 206 mg citric acid, 1.66 g sodium citrate, 140 mg sodium biphosphate, 2 g dextrose, and 17.3 mg adenine). Modified CPD differs from
Transfusion
March-April 1919
standard CPD by containing adenine (0.25 mM final whole blood concentration) and 25 per cent more dextrose. Plasma DEHP Determination
The chemical structure of phthalates reported here was confirmed by using chemical ionization mass spectrometry.' Plasma DEHP concentration was determined by phase extraction of DEHP from plasma followed by quantitation with a Perkin-Elmer 900 gas chromatograph (modification of a procedure communicated privately by J. Miripol of Travenol Laboratories, Chicago, IL). All glassware was thoroughly rinsed in phthalate-free chloroform. Plasma was mixed with 20 volumes of 2: 1 (v/v) ch1oroform:methanol. After equilibration, two volumes of water were added and the phases were allowed to separate. The chloroform phase was collected and dried on a rotary evaporator with water aspiration and 100 pg DNOP in 1 ml CS, was added as internal standard. The columns were 6' by 1 1 8 I.D., 3% OV- 1 on Chromsorb WHP; a nitrogen flow rate of 50 ml per minute was used. The injector, column, and flame ionization detector temperatures were 300,250, and 300 C respectively. Peaks were automatically integrated on a Perkin-Elmer M-l computing integrator. Recovery by this procedure was 81 ? 5 per cent. The lower limit of quantitative sensitivity was 100 ng/ml plasma. All reported values were corrected for the mean recovery of 81 per cent. Plasma MEHP Determination
MEHP assay in plasma was performed by shaking plasma with 3.75 volumes of 2:l (vlv) methano1:chloroform. After centrifugation, the supernatant fluid was saved. The pellet was shaken in 4.75 volumes of 2: 1:0.8 (vlv) methanol: ch1oroform:water for 15 minutes and then centrifuged. The supernatant fluid from the washes were combined. Two and one-half volumes CHCI, and 2.5 volumes of 1 N HCI were added to the supernatant fluid and mixed thoroughly. After phase separation, the chloroform phase was collected, an equal volume of benzene added, and the mixture was evaporated to dryness. The residue was dissolved in one volume of 3:l (v/v) benzene: methanol and the soluble portion was evaporated under nitrogen. The residue was then dissolved in 0.5 ml methanol and 2 ml diethyl ether. Subsequently, 2.5 ml diethyl ether containing = 100 micromoles diazomethane was added and the mixture was allowed to stand for ten minutes prior to nitrogen evaporation, and redissolution in chloroform. Gas chromatographic conditions included: a one meter column of 10% OV-3 on
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DEHP AND MEHP IN BLOOD
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Gas Chrom Z with helium carrier gas at 35 ml/ minute. The temperature was programmed from 140 C to 230 C at a rate of 8 C per minute. The flame ionization detector temperature was 280 C and the injector temperature was 250 C. MEHP was undetectable when DEHP standards were assayed by this procedure. Plastic DEHP and MEHP Determinations Analysis of DEHP and MEHP content in plastic from different bags proceeded as follows: one gram bag shreds were dissolved in 50 ml tetrahydrofuran to which 200 ml CH,OH were added. The mixture was filtered through chloroformwashed #541 filter paper. One ml of filtrate was spiked with 0.5 mg DNOP and dried under nitrogen. The residue was treated with etherial diazomethane (>100 pmoles) and the products were analyzed by gas chromatography on OV-3 columns. Scunning Electron Microscopy In order to provide additional information on morphological differences between the bags, the inner surfaces were studied with the scanning electron microscope. One centimeter square representative sections of plastic from BB-69 and PL-146 blood bags were removed and fastened on studs, in such a way to expose the inner surface of the bags. The specimens were then consecutively coated with carbon, silver, and gold in a Denton Evaporator and photographed through an Etec Autoscan Electron microscope. Calculations Per-unit plasma volume was determined by hernatocrit and prestorage blood volume (by weight and specific gravity) measurements. Statistical differences in plasma DEHP concentrations arising from perturbations inherent in Experiments I and I1 were assessed by subjecting data to a two factor repeated measures analysis of variance Multiple comparisons were made by the method of Dunn.8 The following equation was fitted to the mean plasma DEHP concentrations [DEHP] in Experiments I and I1 at the various sampling times (t) by weighted least-squares. lo .7p**
[DEHP] = [DEHP],
+ ([DEHP], - [DEHP],)e*
The subscripts 0 and m refer to initial and asymptotic [DEHPIs respectively, e is the base of the natural logarithm, and a is the curvilinear accumulation rate. The half-time (TM,) required to reach [DEHP], was computed from TM,
= (In 2)la. The weight used in the regression analysis was the reciprocal of the variance of the mean [DEHP] at each storage time.
Results Experiment I Plasma DEHP concentration (pg/ml) and total plasma DEHP per-unit (mg) in whole blood and red cell concentrates (RCC) (CPD, PI-130) versus storage time are displayed in Figure 1. While DEHP concentration is significantly higher in plasma of RCC than in whole blood plasma at all sampling times after initiation of storage (p < 0.01), the total per-unit plasma DEHP in RCC units ranges from 71 per cent (7th storage day) to 49 per cent (21st storage day) ofthe whole blood per-unit plasma DEHP content. Table 1 shows the results of the curvilinear regression analyses. In PL-130 bags initial [DEHPl0 and asymptotic DEHP concentrations [DEHP], are similar in RCC and whole blood plasmas, but the curvilinear plasma DEHP accumulation rate (a)is 2.86 times greater in RCC units. Experiment I1 Plasma DEHP determinations in modified CPD with adenine stored in PL-146 and BB-69 bags are plotted as concentration (Fg/ml) and per-unit amount (me) in plasma versus storage time in Figure 2. Analysis of variance revealed a significant effect of manufacturer’s bag type on plasma DEHP concentration (p = 0.04) as well as a significant bag typeltime interaction (p = 0.006). This trend of higher DEHP levels with time in blood stored in PL-146 is clearly discernible from 28 to 42 days: mean DEHP levels were 12 to 19 per cent higher in whole blood stored in PL-146 than in BB-69 (p < .05). The curvilinear accumulation rate (a)is 1.8 times greater in whole blood stored in BB-69 relative to blood stored in PL146 bags (Table 1). Mean plasma DEHP and MEHP measurements in 21 and 42 day samples of CPD-adenine blood are shown in Table 2 (columns 3 and 4). DEHP and MEHP concentrations are expressed as pg/ml. MEHP concentrations increased with 4 C storage time and were consistently higher in plasma stored in BB-69 bags than in PL-146 bags. While cumulative concentrations of total phthalates were alike in PL- 146and BB-69, the percentage molar ratio of MEHP to total phthalates was consistently higher in plasma stored in BB-69 (21 days, 14.98 versus 3.74 per cent; 42 days, 17.56 versus 6.29 per cent). The MEHP levels reported in Table 2 were assayed from plasma samples that had been
140
Tnndimion Much-April 1979
PECK ET AL.
2001 175
RCC
WB
FIG.1. Plasma DEHP concentrations (pglml) and DEHP loads(-unit) in red cell concentrates (RCC; n = 3) and whole blood (WB; n = 4) stored in CPD in PL130 bags. The solid lines running through the data points were obtained by fitting the DEHP equation to each data set.
[DEHP] 30
1
A WB
MO/UNIT RCC
7
0
11
14 TIME ( D A Y S )
frozen for 87 to 120 days. PL-146 samples were maintained in frozen storage for four to ten weeks longer than BB-69 samples. One 42-day sample was reanalyzed for MEHP concentration after one year of frozen storage, and this exhibited a 30 per cent (2.42 pB/ml) increase. When 35-day samples were analyzed one year after frozen storage, BB-69 bags also continued to show
consistently higher levels of MEHP than PL-146 (20.8 2 4.2 versus 5.49 f 2.07 pdd; n = 4 each). In Table 3 are the results of the repeat of Expenment I1 in which samples were analyzed for MEHP immediately following their removal from the bag (i.e.,no frozen storage prior to analysis). Plasma MEHP concentrations in BB-69 bags are still consistently higher than in PL-146. Further-
Table 1. Curvilinear Analysis of Plasma DEHP Accumulation During Storage Data Set
PL-130, CPD, RCC, 21 days storage PL-130, CPD, whole blood, 21 days storage PL-146, modified CPD, whole blood, 42 days storage 88-69, modified CPD, whole blood, 42 days storage
[DEHPIo [DEHP], (pglml) (pglml) 5.7 2.3 1.9 3.5
179.0 172.3 295.2 169.3
Q
(Day-') 0.099 0.035 0.016 0.029
TYz, Correlation (days) Coefficient (r) 7.0 19.8 43.3 23.9
0.99 0.99 0.99 0.99
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141
DEHP AND MEHP IN BLOOD 175
1
150
PL-146
125
11-69'A
100
IpG/ML) 75 50
FIG. 2. Plasma DEHP concentrations (pg/ml) and DEHP loads (mg/ unit) in whole blood stored in CPD-adenine in PL-I46(n = 4) and BB-69 (n = 4) bags for 42 days. The solid lines running through the data points were obtained by fitting the DEHP equation to each data set.
25 0
[ DEHP] 60
50
'
PL-146
40
01-69
I.
A '
~(GIUNIT 3 0 20
10
0
0
7
14
21
28
35
42
TIME ( O A Y S )
more, it is apparent that frozen storage itself results in an increase in the plasma MEHP levels in both bags. Experiment Ill
The identity and amounts of phthalates in the plastic bags prior to storage are shown in Table 4. MEHP was not detected in any of the plastics studied.
Grossly and microscopically, the inner surfaces of PL-146 and BB-69 plastics appear dissimilar. When visualized with the scanning electron microscope at low magnification ( 5 3 ~ ) .PL-146 has a smooth surface (Fig. 3), while BB-69 exhibits a surface marked by numerous indentations or pits (Fig. 4). At higher magnification (IWOx), BB-69 exhibits a highly irregular surface with many depressions and elevations both within the
Table 2. MEHP and DEHP Concentrations' at Selected Storage Times (Whole Blood, 4 C) Bag Type
Storage Time
[DEHP] pglml 2 SD
[MEHP] pglml & SD
[Total Phthalateslt pM/ml
PL-146 66-69 PL-146 66-69
21 days 21 days 42 days 42 days
83.1 2 9.1 72.5 2 9.0 152.5 2 4.5 123.4 2 22.4
2.3 0.3 8.8 ? 3.0 6.7 2 3.1 18.7 (n = 1)
0.221 0.216 0.414 0.383
Samples were analyzed following 87-120 days of frozen storage (n = 4).
t [DEHP] + [MEHP].
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PECK ET AL.
Table 3. MEHP Concentrations Measured Immediately Following Removal from Bags (Whole Blood, 4 C)
Bag TVDe
Storage Time
[MEHP] Wglml 2 SD
PL-146 88-69 PL-146 BB-69
21 days 21 days 42 days 42 days
0.1 1.1 4.3 2 0.2 1.82 ? 0.4 12.09 ? 0.2
*
pits and in the surroundingouter surfaces(Fig. 5). In contrast, PL- 146, although also demonstrating an irregular surface, appears to be much flatter (Fig. 6).
Discussion
DEHP Accumulation Contreras et aL5 have shown that DEHP accumulates primarily in plasma of stored blood and that little ( ~ 5 %of total leached DEHP) can be found in erythrocytes. Thus, estimates of potential DEHP loads to recipients of stored blood products may be based on total per-unit DEHP in plasma. The 29 to 51 per cent reduction in total plasma DEHP in a red cell concentrate unit relative to a whole blood unit confirms the report of Miripol and Stern.I3 Based on Experiment I, a table of approximate magnitudes of DEHP exposure to recipients of whole blood and RCC units stored for various time periods at 4 C in PL-130 bags and standard CPD was constructed (Table 5). With increasing storage time, the ratio of per-unit plasma DEHP content in whole blood to that in RCC units becomes larger. Thus, infusion of plasma from one unit of seven day old whole blood delivers 140 per cent of the amount derived from one RCC unit of the same age, whereas at 21 days of storage, one unit of whole blood delivers 204 per cent of the amount obtained from one RCC unit. Therefore, if minimizing exposure to DEHP is desirable, red cell concentrates should be infused when the clinical situation does not specifically require whole blood transfusion. Although total plasma DEHP per unit was considerably less in RCC than in whole
March-April 1979
blood, this finding predominantly reflects the 72 per cent reduction in plasma volume prior to storage. Interestingly, the plasma DEHP concentrationat any point after initiation of storage was not only higher in RCC units than in whole blood units but appears to be reaching its asymptote sooner. This enhanced rate of DEHP accumulation may be related to the increase in ratio of plastic surface area to volume of plasma in RCC units. Throughout 21 days of storage, the ratio of plastic surface area in contact with plasma to total plasma volume (SAIVOL) for whole blood ranged from 1.15 to 1.37, while the SNVOL was 4.08 to 5.94 in RCC units. These estimates take into account variations in donation volume (412 to 444 ml), whole blood hematocrit (0.35 to 0.49, RCC hematocrit (0.68 to 0.74), losses due to sampling, and assumes a constant inner surface in contact with plasma of 391 cm2. Assuming that plasma DEHP accumulation rate is proportional to the S N V O L (massaction law), the rate of DEHP accumulation in RCC units could be postulated to be 2.97 to 5.16 times the accumulation rate in plasma stored in whole blood units. The curvilinear accumulation rate (a)in RCC units was 2.87 times that in whole blood units. Effect of Bug Type und Preservative Whole blood stored for 42 days revealed consistently higher plasma DEHP levels in PL-146 blood bags after 21 days of storage than in BB-69 bags. Comparison of the whole blood DEHP leaching patterns in CPD with CPD-adenine reveals that up to 21 days of storage, concentrations and per-unit totals are similar (Figs. 1 and 2). These data sugTable 4. Phthalate Content of Blood Bag Plastics
PL-130 PL-146 BB-69
303.3 299.6 284.2
mg of phthalate per gram of plastic.
None None None
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DEHP AND MEHP IN BLOOD
143
FIGS.3-6. Top left (3) Low magnification of inner surface of BB-69. Note the numerous depressions or pits in the surface of the plastic ( x 53). Top right (4) Low magnification of the inner surface of PL-146. This plastic appears smooth surfaced in sharp contradistinction to BB-69 ( ~ 5 3 ) .Lower left (5). High magnification of inner surface of BB-69. The plastic demonstrates marked depressions and elevations which occur both inside the pits and in the surrounding outer surfaces ( x 1040). Lower right (6)High magnification of inner surface of PL-146. Although this plastic exhibits an irregular surface, it is considerably flatter than BB-69 ( x 1040).
144
PECK ET AL.
Table 5. Approximate' Load (mg) to Patients of DEHP Contained in Plasma Days of Storage
~
Number of Units
7
14
21
Whole Blood 1 3 6
12 36 72
20 60 120
27 81 162
Red Cell Concentrate 1 9 3 27 54 6
10 30 60
13 39 78
~~
Rounded to the nearest mg.
gest that the presence of adenine in CPD does not strongly influence DEHP leachingpatterns. Curvilinear DEHP Accumulation The general shape of the cumulative DEHP concentration time plots is curvilinear. However, other investigator^^*'^ have fit a straight line to phthalate accumulation data, even though visual examination of the resulting plot and evaluation of the correlation coefficient would not appear to warrant this interpretation. As an alternative approach to examining plasma DEHP accumulation data, we fit the DEHP equation to the plasma DEHP data derived from Experiments I and 11. This equation is intended as a summarizing technique and simultaneously estimates the initial and asymptotic plasma DEHP concentrations as well as a first order rate constant (a).The curvilinear accumulation rate (a)can be used to evaluate hypotheses regkding possible influencing variables, such as the SNVOL. The half time (Tl12,)to reach [DEHP], can be computed and is a useful way of evaluating the rate of DEHP accumulation. Source of MEHP Analysis of the phthalate composition of plastic strips cut from the blood bags clearly established that MEHP did not originate from the PVC plastic of any of the bags investigated. Instead, MEHP in plasma
Tranlfusion March-April 1979
arises from conversion of DEHP to MEHP during storage. Human plasma is known to contain nonspecific lipasese which may catalyze the cleavage of 2-ethylhexanol from one of the carbonyl carbons connecting it to the benzene ring of DEHP, thereby producing MEHP. In support of this hypothesis, Albro and Thomas* have shown that DEHP can be hydrolyzed to MEHP by lipases from a variety of rat tissues including plasma lipoprotein-lipase. Although 2ethylhexanol concentration was not measured in our studies, it is reasonable to postulate that the conversion of DEHP to MEHP during storage results in the accumulation of this product also, providing it is not further metabolized. Moreover, further hydrolysis of MEHP to phthalic acid is also possible, but was not studied in this series of experiments. It is unclear why MEHP accumulation in whole blood stored in BB-69 bags was higher than in PL-146 bags. After 21 and 42 days storage, the total phthalates leached into plasma is only slightly less in BB-69 than in PL-146. Thus, the higher plasma MEHP and lower DEHP concentrations in the BB-69 bags appear related to an increased conversion of DEHP to MEHP. This finding suggests an enhanced lipase activity in the BB-69 bags. Conceivably, differences in the surface characteristics of the two plastics could account for the postulated differences in lipase activity. Lipase is known to adsorb to hydrophobic surfaces including teflon powder and polystyrene latex beads.430 Such adsorption causes an enhancement of lipolytic activity of up to three orders of magnitude and the augmentation appears directly related to the added surface area.20Clearly, there is a marked difference in the surface characteristics of the two blood storage containers, PL-146 and BB-69. The pitted, rough surface of BB-69 provides a larger surface area than that of the smoother PL-146. The observation that the DEHP curvilinear accumulation rate (a)in BB-69 is 1.76 times that in PL-146 is consistent with an aug-
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DEHP AND MEHP IN BLOOD
mented rate of DEHP migration into plasma due to increased SNVOL. Whether this difference in surface topography between the two types of plastic also contributed to the enhanced MEHP accumulation in BB-69 bags cannot be answered by the present investigation. Plasma MEHP During Frozen Storage The 30 per cent increase in plasma MEHP concentration found in the sample which was reanalyzed following one year of frozen storage suggested that DEHP continues to be hydrolyzed even in the frozen state. This interpretation is further supported by the 35 day BB-69 samples analyzed following one year of frozen storage which showed a mean MEHP concentration higher than that of 42 day samples frozen for only 87 days. Finally, the repeat of Experiment I1 yielded lower mean MEHP values when samples were analyzed immediately after removal from bags than those in the original Experiment I1 which had been frozen 87 to 120days prior to analysis. SigniJicance of MEHP The detection of MEHP in this series of experiments is not an isolated observation. We have measured MEHP concentrations of 16.4 pg/ml in platelet concentrate stored for 50 hours at 22 C.15 Rubinl9 reported finding = 14.0pg/ml MEHP in plasma stored in PL-130 bags for 21 days at 4 C and 55.5 pg/ml in plasma incubated with PL-130 for 24 hours at 37 C. The toxicologic significance of the presence of MEHP (and possibly 2-ethylhexanol) in transfusion blood and blood products is unknown. Peterson" reported LD 50's ranging from 0.54 to 1.5 m moleslkg in rats and mice following intravenous or intraperitoneal infusion of MEHP dissolved in 5% NaHCO,. This dosing formulation was noted to be characterized by severe local toxicity. Lake et al." reported that MEHP or 2-ethylhexanol (5 m moles/kg/day) in the diet of rats each resulted in apparent hepatotoxicity after seven days. The evidence for liver damage
was similar to that caused by dietary DEHP at comparable dose levels. However, such massive exposure (equivalent to seven daily human infusions of 490 to 1900 unitslday of 21 day old blood), given orally in a nonprimate species, bears uncertain relevance to humans whose parenteral exposure to DEHP and MEHP is much lower. Indeed, the exact exposure mode ( e . g . , route of administration and physical state of the dosing formulation) has been shown to influence the pharmacology and toxicology of phthalates .6 These experiments establish that : 1) plasma of red cell concentrates stored in PL-130 contains less total DEHP per unit than plasma of whole blood units in PL-130; 2) DEHP accumulation in plasma during storage is a curvilinear phenomenon; 3) MEHP accumulates in plasma during storage and continues to accumulate in frozen plasma samples; 4) the MEHP accumulation rate is apparently affected by the manufacturer-source of the bags; and 5 ) no MEHP can be found in the plastic of PVC blood containers. The clinical significance of these facts awaits further information concerning DEHP and MEHP toxicity in humans exposed through transfusion of stored blood products. Acknowledgments The authors wish to express gratitude to Mr. Alan Hopkins Specialist Berton Barrett for assistance in statistical analyses and to Ms. Celeste Mangold and Mrs. Lottie Applewhite for their aid in manuscript preparation and technical editing.
References Albro, P. W., and B. Moore: Identification of the metabolites of simple phthalate diesters in rat urine. J. Chromatogr. 94:209, 1974. -, and R. 0. Thomas: Enzymatic hydrolysis of di~2ethylhexyl)phthalateby lipases. Biochim. Biophys. Acta W.380, 1973. Autian, J.: Toxicity and health threats of phthalate esters: review of the literature. Environ. Health Perspect. 4:3, 1973. Brockman, H. L., J . H. Law, and F. J . Kezdy: Catalysis by adsorbed enzymes. J. Biol. Chem. 248:4%5, 1973.
PECK ET AL. 5. Contreras, T. J., R. H. Sheibley, and C. R. Valeri:
6.
7. 8. 9. 10.
11.
12. 13.
14.
IS.
16.
Afcumulation of di-2-ethylhexyl phthalate (DEHP) in whole blood, platelet concentrates, and platelet-poor plasma. Transfusion 1434, 1974. Darby, T. D., and R. F. Wallin: Some quantitative aspects of toxicity. Pharmacologist 18171, 1976. Dixon, W. J.: BMDP Biomedical Computer Programs, (BMDPZV). Berkeley, University of California Press, 1975. Dunn, 0. J.: Multiple comparisons among means. J. Am. Stat. Assn. 5652, 1961. Fishman, W.H.: Plasma Enzymes. In:The Plasma Roteins, vol. 2. F. W. Putnam, Ed. New York, Academic Press, 1960, p 70. Horowitz, D. L., and L. D. Hoover: Nonlinear parameter estimation. 11. Analysis of Biomedical Data by Time Sharing. I. Nonlinear Regression Analysis, Naval Medical Research Institute Report No. 25, Project No. MR005.20-0287, 26 February 1970; modified by C. Peck and B. Barrett, Letterman Army Institute of Research, Presidio of San Francisco, CA, 1976. Lake, B. G., S. D. Gangolli, P. Grasso, and A. G. Lloyd: Studies on the hepatic effects of orally administered di-(2-ethylhexyl) phthalate in the rat. Toxicol. Appl. Pharmacol. 32355, 1975. Miller, W. V., Ed.: Technical Methods and Procedures of the American Association of Blood Banks, 6th ed. Washington, D.C., 1974. Miripol, J. E., andI. J. Stem: Decreasedaccumulation of phthalate plasticizer during storage of blood as packed cells. Transfusion 17:71. 1977. , P. J. Garvin, and R. F. Wallin: Contract NOI-HB-22990 Final Technical Progress Report: Role of lipids, plasma protein concentration and plasma lipoprotein concentration on DEHP accumulation in plasma. Travenol Laboratories, Morton Grove, Ill., September 1975, pp. 4. Peck, C. C.. and P. W. Albro: Letterman Army Institute of Research Notebook No. 5-302.1, Letterman Army Institute of Research, Presidio of San Francisco. CA, Nov. 10, 1976. page 95. ,and T. F. Zuck: Disposition and metabolism of DEHP in primates. Workshop on Adenine and Red Cell Preservation. In: Transcript of
-
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Transfusion MWh-AMI 1579
Proceedings: Dept. Health, Education and Welfare, Food and Drug Administration, Bureau of Biologics, Washington, D.C., 1 October 1976. 17. Peterson, R. V.: Toxicology of plastic devices having contact with blood. Final Report, N.I.H. Contract NIH-NHLI-73-2908-B:35-41, 1975. 18. The Pharmacopeiaof the United States of America, 18th Revision, Anticoagulant Citrate Phosphate Dextrose Solution. Easton, PA, Mack Printing Co., 1970, p. 47. 19. Rubin, R. T.: DEHP leaching and toxicity. Workshop on Adenine and Red Cell Reservation. Transcript of Proceedings: Dept. Health, Education and Welfare, Food and Drug Administration, Bureau of Biologics, Washington, D.C. 20. Suguira, M..and M. Isobe: Studies on the mechanism of lipase reaction. 111. Adsorption of chromobacterium lipase on hydrophobic glass beads. Chem. Pharm. Bull. 24.72, 1976. 21. Swisher, S. N.: The introduction of adenine fortified blood preservatives: Introduction and an interpretation of its history. Transfusion 17:309, 1977. 22. Winer, B. J.: Statistical Principles in Experimental Design. New York, McGraw-Hill, 1962, p. 518. p. 518.
Carl C. Peck, M.D.. LTC. MC, Division of Blood Research, Letterman Army Institute of Research, Presidio of San Francisco. Daniel G. Odom, Ph.D., Cornell College, Mount Vernon, Iowa. Harold I. Friedman, M.D., Ph.D.. Department of Surgery, Health Science Center, University of Arizona. Tucson, Arizona. Phillip W. Albro, Ph.D., National Institutes of Environmental Health Sciences, Research Triangle Park, North Carolina. J. Ronald Hass, Ph.D., National Institutes of Environmental Health Sciences. John T. Brady. University of Colorado Medical Center, Denver, Colorado. SPS Don A. Jess, B.S.. Physical Sciences Assistant, Division of Blood Research, Letterman Army Institute of Research, Presidio of San Francisco.
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Plasma DEHP concentrations w e n measured weekly la whok bloodand red Ccn concentrates(RCC) during 21 days of storage in standard CPD within PL-130 blood bags. In addition, DEHP and MEHP accumulation pattermwere lnvest@td in blood s t d for 42 days in modiAed CPD with adenine wlthin PL-146 and BB-69 storage containers. Total per-unit plasma DEHP of RCC units was 49 to 71 percea of the total In plasma of whok bbod units (PL.130). From 28 to 42 days, IIRM DEHP levels were 12 to 19 per cent higher in whole blood stored In PL-146 than in B W . Although MEHP was aot found in m y blood bag plastic, MEHP accumulated in plasma during whok blood storage. MEHP concentrations were 2.8 to 3.8 times higher In plesnu StOFed In BB49 than h PL-146. It is postulatedthat MEHP a r k from hydrolysis of DEHP by pinsma lipase, even in frozen plasma Mmplcs, and th.t tk n te or this renctbn is -ni by blood bag piastk surface characteristks.
INFUSION of the plasticizer DEHP leached from polyvinylchloride (PVC) bags into blood during cold storage has been considered a possible health hazard.3 To assess the po- tential magnitude of human exposure to this compound, a knowledge of the quantity of DEHP present in units of blood or red cell concentrate after given storage periods is desirable. In the United States, blood and blood products are stored in PVC bags marketed by two manufacturers, but DEHP leaching patterns have been reported only Received for publication February 6 , 1978; accepted March 5 , 1978. The opinions of assertions contained herein are the views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or Department of Defense.
137
for blood stored in one manufacturer's bag.s*13Furthermore, little is known concerning DEHP accumulation in blood preserved in adenine-enriched CPD during extended storage (greater than 21 days). This latter problem is of considerable interest since licensure of adenine fortified blood is being considered.z1 In studies on the metabolic fate of DEHP in primates (African Green Monkey and man), MEHP and oxidation products of MEHP were found in plasma and urine following intravenous administrationof plasma containing leached DEHP.16 While the MEHP compounds were derived primarily from in vivo metabolism of DEHP, small concentrations of MEHP were discovered in the infusion plasmas. This latter finding prompted a search for MEHP in the plastic of PVC blood containers currently in use as well as in plasma during storage. The present investigation was designed to provide the following information: 1) DEHP accumulation in plasma of whole blood and red cell concentrates stored in CPD for three weeks at 4 C in one type of blood storage container; 2) DEHP and MEHP accumulation in blood in PVC containers from two different manufacturers kept for six weeks at 4 C but preserved in adenine-fortified CPD, 3) DEHP and MEHP content in the plastic of unused blood bags from the same two manufacturers; and, 4) the source and accumulationpattern of MEHPduring storage.
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PECK ET AL.
Methods Two blood bag manufacturers fabricated three types of plastic storage containers utilized in this study: PL-130, PL-146 (Fenwal Laboratories, Morton Grove, IL), and BB-69 (McGaw Laboratories, Santa Ana. CA). In Experiment I, the accumulation of DEHP in plasma was determined and compared in four whole blood and four red cell concentrate units (average Hct = 71%), stored in standard CPDI8 for 21 days within PL130 blood bags. In Experiment 11, plasma DEHP and MEHP accumulation were measured in blood stored for 42 days in adenine-enriched CPD within PL-146 and BB-69 blood bags. In both Experiments I and 11, each unit was mixed, sampled (10 ml whole blood) aseptically at weekly intervals, centrifuged, and the plasma was frozen in phthalate free glassware for later analysis. After 87 to 120 days of frozen storage, plasma DEHP concentrations were measured in all samples. Following an identical frozen storage period, plasma MEHP concentrations were measured in 21 and 42 day samples from Experiment 11. In order to assess possible MEHP accumulation during frozen storage (-25 C), one 42-day sample (BB-69) was reanalyzed after 360 days of frozen storage. In addition, 35-day samples from the same experiment which had been frozen for one year were analyzed for MEHP concentration. Finally, Experiment I1 was repeated in two PL-146 and two BB-69 bags, and the samples were analyzed for MEHP immediately after removal from the bag on storage days 21 and 42. After termination of each experiment, the contents of blood bags were cultured. Bacterial contamination was noted in only one case: a red cell concentrate unit in Experiment I, the results of which were discarded. In Experiment 111, phthalate identity and content were determined in strips of PL- 130, PL- 146, and BB-69 plastics cut from unused bags. The inner surfaces (side in contact with blood during storage) of identical strips were subjected to electron microscopic examination. Blood Collection and Storage
One unit of blood was drawn from each of 16 healthy adults who had given voluntary informed consent. The units were stored at 4 C within 30 minutes of phlebotomy.12Red cell concentrate units were centrifuged to a hematocrit of =70 per cent. Two anticoagulants were used: 1) standard citrate-phosphatedextrose(CPD)I8and 2) modified CPD (per 63 ml: 206 mg citric acid, 1.66 g sodium citrate, 140 mg sodium biphosphate, 2 g dextrose, and 17.3 mg adenine). Modified CPD differs from
Transfusion
March-April 1919
standard CPD by containing adenine (0.25 mM final whole blood concentration) and 25 per cent more dextrose. Plasma DEHP Determination
The chemical structure of phthalates reported here was confirmed by using chemical ionization mass spectrometry.' Plasma DEHP concentration was determined by phase extraction of DEHP from plasma followed by quantitation with a Perkin-Elmer 900 gas chromatograph (modification of a procedure communicated privately by J. Miripol of Travenol Laboratories, Chicago, IL). All glassware was thoroughly rinsed in phthalate-free chloroform. Plasma was mixed with 20 volumes of 2: 1 (v/v) ch1oroform:methanol. After equilibration, two volumes of water were added and the phases were allowed to separate. The chloroform phase was collected and dried on a rotary evaporator with water aspiration and 100 pg DNOP in 1 ml CS, was added as internal standard. The columns were 6' by 1 1 8 I.D., 3% OV- 1 on Chromsorb WHP; a nitrogen flow rate of 50 ml per minute was used. The injector, column, and flame ionization detector temperatures were 300,250, and 300 C respectively. Peaks were automatically integrated on a Perkin-Elmer M-l computing integrator. Recovery by this procedure was 81 ? 5 per cent. The lower limit of quantitative sensitivity was 100 ng/ml plasma. All reported values were corrected for the mean recovery of 81 per cent. Plasma MEHP Determination
MEHP assay in plasma was performed by shaking plasma with 3.75 volumes of 2:l (vlv) methano1:chloroform. After centrifugation, the supernatant fluid was saved. The pellet was shaken in 4.75 volumes of 2: 1:0.8 (vlv) methanol: ch1oroform:water for 15 minutes and then centrifuged. The supernatant fluid from the washes were combined. Two and one-half volumes CHCI, and 2.5 volumes of 1 N HCI were added to the supernatant fluid and mixed thoroughly. After phase separation, the chloroform phase was collected, an equal volume of benzene added, and the mixture was evaporated to dryness. The residue was dissolved in one volume of 3:l (v/v) benzene: methanol and the soluble portion was evaporated under nitrogen. The residue was then dissolved in 0.5 ml methanol and 2 ml diethyl ether. Subsequently, 2.5 ml diethyl ether containing = 100 micromoles diazomethane was added and the mixture was allowed to stand for ten minutes prior to nitrogen evaporation, and redissolution in chloroform. Gas chromatographic conditions included: a one meter column of 10% OV-3 on
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DEHP AND MEHP IN BLOOD
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Gas Chrom Z with helium carrier gas at 35 ml/ minute. The temperature was programmed from 140 C to 230 C at a rate of 8 C per minute. The flame ionization detector temperature was 280 C and the injector temperature was 250 C. MEHP was undetectable when DEHP standards were assayed by this procedure. Plastic DEHP and MEHP Determinations Analysis of DEHP and MEHP content in plastic from different bags proceeded as follows: one gram bag shreds were dissolved in 50 ml tetrahydrofuran to which 200 ml CH,OH were added. The mixture was filtered through chloroformwashed #541 filter paper. One ml of filtrate was spiked with 0.5 mg DNOP and dried under nitrogen. The residue was treated with etherial diazomethane (>100 pmoles) and the products were analyzed by gas chromatography on OV-3 columns. Scunning Electron Microscopy In order to provide additional information on morphological differences between the bags, the inner surfaces were studied with the scanning electron microscope. One centimeter square representative sections of plastic from BB-69 and PL-146 blood bags were removed and fastened on studs, in such a way to expose the inner surface of the bags. The specimens were then consecutively coated with carbon, silver, and gold in a Denton Evaporator and photographed through an Etec Autoscan Electron microscope. Calculations Per-unit plasma volume was determined by hernatocrit and prestorage blood volume (by weight and specific gravity) measurements. Statistical differences in plasma DEHP concentrations arising from perturbations inherent in Experiments I and I1 were assessed by subjecting data to a two factor repeated measures analysis of variance Multiple comparisons were made by the method of Dunn.8 The following equation was fitted to the mean plasma DEHP concentrations [DEHP] in Experiments I and I1 at the various sampling times (t) by weighted least-squares. lo .7p**
[DEHP] = [DEHP],
+ ([DEHP], - [DEHP],)e*
The subscripts 0 and m refer to initial and asymptotic [DEHPIs respectively, e is the base of the natural logarithm, and a is the curvilinear accumulation rate. The half-time (TM,) required to reach [DEHP], was computed from TM,
= (In 2)la. The weight used in the regression analysis was the reciprocal of the variance of the mean [DEHP] at each storage time.
Results Experiment I Plasma DEHP concentration (pg/ml) and total plasma DEHP per-unit (mg) in whole blood and red cell concentrates (RCC) (CPD, PI-130) versus storage time are displayed in Figure 1. While DEHP concentration is significantly higher in plasma of RCC than in whole blood plasma at all sampling times after initiation of storage (p < 0.01), the total per-unit plasma DEHP in RCC units ranges from 71 per cent (7th storage day) to 49 per cent (21st storage day) ofthe whole blood per-unit plasma DEHP content. Table 1 shows the results of the curvilinear regression analyses. In PL-130 bags initial [DEHPl0 and asymptotic DEHP concentrations [DEHP], are similar in RCC and whole blood plasmas, but the curvilinear plasma DEHP accumulation rate (a)is 2.86 times greater in RCC units. Experiment I1 Plasma DEHP determinations in modified CPD with adenine stored in PL-146 and BB-69 bags are plotted as concentration (Fg/ml) and per-unit amount (me) in plasma versus storage time in Figure 2. Analysis of variance revealed a significant effect of manufacturer’s bag type on plasma DEHP concentration (p = 0.04) as well as a significant bag typeltime interaction (p = 0.006). This trend of higher DEHP levels with time in blood stored in PL-146 is clearly discernible from 28 to 42 days: mean DEHP levels were 12 to 19 per cent higher in whole blood stored in PL-146 than in BB-69 (p < .05). The curvilinear accumulation rate (a)is 1.8 times greater in whole blood stored in BB-69 relative to blood stored in PL146 bags (Table 1). Mean plasma DEHP and MEHP measurements in 21 and 42 day samples of CPD-adenine blood are shown in Table 2 (columns 3 and 4). DEHP and MEHP concentrations are expressed as pg/ml. MEHP concentrations increased with 4 C storage time and were consistently higher in plasma stored in BB-69 bags than in PL-146 bags. While cumulative concentrations of total phthalates were alike in PL- 146and BB-69, the percentage molar ratio of MEHP to total phthalates was consistently higher in plasma stored in BB-69 (21 days, 14.98 versus 3.74 per cent; 42 days, 17.56 versus 6.29 per cent). The MEHP levels reported in Table 2 were assayed from plasma samples that had been
140
Tnndimion Much-April 1979
PECK ET AL.
2001 175
RCC
WB
FIG.1. Plasma DEHP concentrations (pglml) and DEHP loads(-unit) in red cell concentrates (RCC; n = 3) and whole blood (WB; n = 4) stored in CPD in PL130 bags. The solid lines running through the data points were obtained by fitting the DEHP equation to each data set.
[DEHP] 30
1
A WB
MO/UNIT RCC
7
0
11
14 TIME ( D A Y S )
frozen for 87 to 120 days. PL-146 samples were maintained in frozen storage for four to ten weeks longer than BB-69 samples. One 42-day sample was reanalyzed for MEHP concentration after one year of frozen storage, and this exhibited a 30 per cent (2.42 pB/ml) increase. When 35-day samples were analyzed one year after frozen storage, BB-69 bags also continued to show
consistently higher levels of MEHP than PL-146 (20.8 2 4.2 versus 5.49 f 2.07 pdd; n = 4 each). In Table 3 are the results of the repeat of Expenment I1 in which samples were analyzed for MEHP immediately following their removal from the bag (i.e.,no frozen storage prior to analysis). Plasma MEHP concentrations in BB-69 bags are still consistently higher than in PL-146. Further-
Table 1. Curvilinear Analysis of Plasma DEHP Accumulation During Storage Data Set
PL-130, CPD, RCC, 21 days storage PL-130, CPD, whole blood, 21 days storage PL-146, modified CPD, whole blood, 42 days storage 88-69, modified CPD, whole blood, 42 days storage
[DEHPIo [DEHP], (pglml) (pglml) 5.7 2.3 1.9 3.5
179.0 172.3 295.2 169.3
Q
(Day-') 0.099 0.035 0.016 0.029
TYz, Correlation (days) Coefficient (r) 7.0 19.8 43.3 23.9
0.99 0.99 0.99 0.99
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141
DEHP AND MEHP IN BLOOD 175
1
150
PL-146
125
11-69'A
100
IpG/ML) 75 50
FIG. 2. Plasma DEHP concentrations (pg/ml) and DEHP loads (mg/ unit) in whole blood stored in CPD-adenine in PL-I46(n = 4) and BB-69 (n = 4) bags for 42 days. The solid lines running through the data points were obtained by fitting the DEHP equation to each data set.
25 0
[ DEHP] 60
50
'
PL-146
40
01-69
I.
A '
~(GIUNIT 3 0 20
10
0
0
7
14
21
28
35
42
TIME ( O A Y S )
more, it is apparent that frozen storage itself results in an increase in the plasma MEHP levels in both bags. Experiment Ill
The identity and amounts of phthalates in the plastic bags prior to storage are shown in Table 4. MEHP was not detected in any of the plastics studied.
Grossly and microscopically, the inner surfaces of PL-146 and BB-69 plastics appear dissimilar. When visualized with the scanning electron microscope at low magnification ( 5 3 ~ ) .PL-146 has a smooth surface (Fig. 3), while BB-69 exhibits a surface marked by numerous indentations or pits (Fig. 4). At higher magnification (IWOx), BB-69 exhibits a highly irregular surface with many depressions and elevations both within the
Table 2. MEHP and DEHP Concentrations' at Selected Storage Times (Whole Blood, 4 C) Bag Type
Storage Time
[DEHP] pglml 2 SD
[MEHP] pglml & SD
[Total Phthalateslt pM/ml
PL-146 66-69 PL-146 66-69
21 days 21 days 42 days 42 days
83.1 2 9.1 72.5 2 9.0 152.5 2 4.5 123.4 2 22.4
2.3 0.3 8.8 ? 3.0 6.7 2 3.1 18.7 (n = 1)
0.221 0.216 0.414 0.383
Samples were analyzed following 87-120 days of frozen storage (n = 4).
t [DEHP] + [MEHP].
142
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PECK ET AL.
Table 3. MEHP Concentrations Measured Immediately Following Removal from Bags (Whole Blood, 4 C)
Bag TVDe
Storage Time
[MEHP] Wglml 2 SD
PL-146 88-69 PL-146 BB-69
21 days 21 days 42 days 42 days
0.1 1.1 4.3 2 0.2 1.82 ? 0.4 12.09 ? 0.2
*
pits and in the surroundingouter surfaces(Fig. 5). In contrast, PL- 146, although also demonstrating an irregular surface, appears to be much flatter (Fig. 6).
Discussion
DEHP Accumulation Contreras et aL5 have shown that DEHP accumulates primarily in plasma of stored blood and that little ( ~ 5 %of total leached DEHP) can be found in erythrocytes. Thus, estimates of potential DEHP loads to recipients of stored blood products may be based on total per-unit DEHP in plasma. The 29 to 51 per cent reduction in total plasma DEHP in a red cell concentrate unit relative to a whole blood unit confirms the report of Miripol and Stern.I3 Based on Experiment I, a table of approximate magnitudes of DEHP exposure to recipients of whole blood and RCC units stored for various time periods at 4 C in PL-130 bags and standard CPD was constructed (Table 5). With increasing storage time, the ratio of per-unit plasma DEHP content in whole blood to that in RCC units becomes larger. Thus, infusion of plasma from one unit of seven day old whole blood delivers 140 per cent of the amount derived from one RCC unit of the same age, whereas at 21 days of storage, one unit of whole blood delivers 204 per cent of the amount obtained from one RCC unit. Therefore, if minimizing exposure to DEHP is desirable, red cell concentrates should be infused when the clinical situation does not specifically require whole blood transfusion. Although total plasma DEHP per unit was considerably less in RCC than in whole
March-April 1979
blood, this finding predominantly reflects the 72 per cent reduction in plasma volume prior to storage. Interestingly, the plasma DEHP concentrationat any point after initiation of storage was not only higher in RCC units than in whole blood units but appears to be reaching its asymptote sooner. This enhanced rate of DEHP accumulation may be related to the increase in ratio of plastic surface area to volume of plasma in RCC units. Throughout 21 days of storage, the ratio of plastic surface area in contact with plasma to total plasma volume (SAIVOL) for whole blood ranged from 1.15 to 1.37, while the SNVOL was 4.08 to 5.94 in RCC units. These estimates take into account variations in donation volume (412 to 444 ml), whole blood hematocrit (0.35 to 0.49, RCC hematocrit (0.68 to 0.74), losses due to sampling, and assumes a constant inner surface in contact with plasma of 391 cm2. Assuming that plasma DEHP accumulation rate is proportional to the S N V O L (massaction law), the rate of DEHP accumulation in RCC units could be postulated to be 2.97 to 5.16 times the accumulation rate in plasma stored in whole blood units. The curvilinear accumulation rate (a)in RCC units was 2.87 times that in whole blood units. Effect of Bug Type und Preservative Whole blood stored for 42 days revealed consistently higher plasma DEHP levels in PL-146 blood bags after 21 days of storage than in BB-69 bags. Comparison of the whole blood DEHP leaching patterns in CPD with CPD-adenine reveals that up to 21 days of storage, concentrations and per-unit totals are similar (Figs. 1 and 2). These data sugTable 4. Phthalate Content of Blood Bag Plastics
PL-130 PL-146 BB-69
303.3 299.6 284.2
mg of phthalate per gram of plastic.
None None None
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DEHP AND MEHP IN BLOOD
143
FIGS.3-6. Top left (3) Low magnification of inner surface of BB-69. Note the numerous depressions or pits in the surface of the plastic ( x 53). Top right (4) Low magnification of the inner surface of PL-146. This plastic appears smooth surfaced in sharp contradistinction to BB-69 ( ~ 5 3 ) .Lower left (5). High magnification of inner surface of BB-69. The plastic demonstrates marked depressions and elevations which occur both inside the pits and in the surrounding outer surfaces ( x 1040). Lower right (6)High magnification of inner surface of PL-146. Although this plastic exhibits an irregular surface, it is considerably flatter than BB-69 ( x 1040).
144
PECK ET AL.
Table 5. Approximate' Load (mg) to Patients of DEHP Contained in Plasma Days of Storage
~
Number of Units
7
14
21
Whole Blood 1 3 6
12 36 72
20 60 120
27 81 162
Red Cell Concentrate 1 9 3 27 54 6
10 30 60
13 39 78
~~
Rounded to the nearest mg.
gest that the presence of adenine in CPD does not strongly influence DEHP leachingpatterns. Curvilinear DEHP Accumulation The general shape of the cumulative DEHP concentration time plots is curvilinear. However, other investigator^^*'^ have fit a straight line to phthalate accumulation data, even though visual examination of the resulting plot and evaluation of the correlation coefficient would not appear to warrant this interpretation. As an alternative approach to examining plasma DEHP accumulation data, we fit the DEHP equation to the plasma DEHP data derived from Experiments I and 11. This equation is intended as a summarizing technique and simultaneously estimates the initial and asymptotic plasma DEHP concentrations as well as a first order rate constant (a).The curvilinear accumulation rate (a)can be used to evaluate hypotheses regkding possible influencing variables, such as the SNVOL. The half time (Tl12,)to reach [DEHP], can be computed and is a useful way of evaluating the rate of DEHP accumulation. Source of MEHP Analysis of the phthalate composition of plastic strips cut from the blood bags clearly established that MEHP did not originate from the PVC plastic of any of the bags investigated. Instead, MEHP in plasma
Tranlfusion March-April 1979
arises from conversion of DEHP to MEHP during storage. Human plasma is known to contain nonspecific lipasese which may catalyze the cleavage of 2-ethylhexanol from one of the carbonyl carbons connecting it to the benzene ring of DEHP, thereby producing MEHP. In support of this hypothesis, Albro and Thomas* have shown that DEHP can be hydrolyzed to MEHP by lipases from a variety of rat tissues including plasma lipoprotein-lipase. Although 2ethylhexanol concentration was not measured in our studies, it is reasonable to postulate that the conversion of DEHP to MEHP during storage results in the accumulation of this product also, providing it is not further metabolized. Moreover, further hydrolysis of MEHP to phthalic acid is also possible, but was not studied in this series of experiments. It is unclear why MEHP accumulation in whole blood stored in BB-69 bags was higher than in PL-146 bags. After 21 and 42 days storage, the total phthalates leached into plasma is only slightly less in BB-69 than in PL-146. Thus, the higher plasma MEHP and lower DEHP concentrations in the BB-69 bags appear related to an increased conversion of DEHP to MEHP. This finding suggests an enhanced lipase activity in the BB-69 bags. Conceivably, differences in the surface characteristics of the two plastics could account for the postulated differences in lipase activity. Lipase is known to adsorb to hydrophobic surfaces including teflon powder and polystyrene latex beads.430 Such adsorption causes an enhancement of lipolytic activity of up to three orders of magnitude and the augmentation appears directly related to the added surface area.20Clearly, there is a marked difference in the surface characteristics of the two blood storage containers, PL-146 and BB-69. The pitted, rough surface of BB-69 provides a larger surface area than that of the smoother PL-146. The observation that the DEHP curvilinear accumulation rate (a)in BB-69 is 1.76 times that in PL-146 is consistent with an aug-
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DEHP AND MEHP IN BLOOD
mented rate of DEHP migration into plasma due to increased SNVOL. Whether this difference in surface topography between the two types of plastic also contributed to the enhanced MEHP accumulation in BB-69 bags cannot be answered by the present investigation. Plasma MEHP During Frozen Storage The 30 per cent increase in plasma MEHP concentration found in the sample which was reanalyzed following one year of frozen storage suggested that DEHP continues to be hydrolyzed even in the frozen state. This interpretation is further supported by the 35 day BB-69 samples analyzed following one year of frozen storage which showed a mean MEHP concentration higher than that of 42 day samples frozen for only 87 days. Finally, the repeat of Experiment I1 yielded lower mean MEHP values when samples were analyzed immediately after removal from bags than those in the original Experiment I1 which had been frozen 87 to 120days prior to analysis. SigniJicance of MEHP The detection of MEHP in this series of experiments is not an isolated observation. We have measured MEHP concentrations of 16.4 pg/ml in platelet concentrate stored for 50 hours at 22 C.15 Rubinl9 reported finding = 14.0pg/ml MEHP in plasma stored in PL-130 bags for 21 days at 4 C and 55.5 pg/ml in plasma incubated with PL-130 for 24 hours at 37 C. The toxicologic significance of the presence of MEHP (and possibly 2-ethylhexanol) in transfusion blood and blood products is unknown. Peterson" reported LD 50's ranging from 0.54 to 1.5 m moleslkg in rats and mice following intravenous or intraperitoneal infusion of MEHP dissolved in 5% NaHCO,. This dosing formulation was noted to be characterized by severe local toxicity. Lake et al." reported that MEHP or 2-ethylhexanol (5 m moles/kg/day) in the diet of rats each resulted in apparent hepatotoxicity after seven days. The evidence for liver damage
was similar to that caused by dietary DEHP at comparable dose levels. However, such massive exposure (equivalent to seven daily human infusions of 490 to 1900 unitslday of 21 day old blood), given orally in a nonprimate species, bears uncertain relevance to humans whose parenteral exposure to DEHP and MEHP is much lower. Indeed, the exact exposure mode ( e . g . , route of administration and physical state of the dosing formulation) has been shown to influence the pharmacology and toxicology of phthalates .6 These experiments establish that : 1) plasma of red cell concentrates stored in PL-130 contains less total DEHP per unit than plasma of whole blood units in PL-130; 2) DEHP accumulation in plasma during storage is a curvilinear phenomenon; 3) MEHP accumulates in plasma during storage and continues to accumulate in frozen plasma samples; 4) the MEHP accumulation rate is apparently affected by the manufacturer-source of the bags; and 5 ) no MEHP can be found in the plastic of PVC blood containers. The clinical significance of these facts awaits further information concerning DEHP and MEHP toxicity in humans exposed through transfusion of stored blood products. Acknowledgments The authors wish to express gratitude to Mr. Alan Hopkins Specialist Berton Barrett for assistance in statistical analyses and to Ms. Celeste Mangold and Mrs. Lottie Applewhite for their aid in manuscript preparation and technical editing.
References Albro, P. W., and B. Moore: Identification of the metabolites of simple phthalate diesters in rat urine. J. Chromatogr. 94:209, 1974. -, and R. 0. Thomas: Enzymatic hydrolysis of di~2ethylhexyl)phthalateby lipases. Biochim. Biophys. Acta W.380, 1973. Autian, J.: Toxicity and health threats of phthalate esters: review of the literature. Environ. Health Perspect. 4:3, 1973. Brockman, H. L., J . H. Law, and F. J . Kezdy: Catalysis by adsorbed enzymes. J. Biol. Chem. 248:4%5, 1973.
PECK ET AL. 5. Contreras, T. J., R. H. Sheibley, and C. R. Valeri:
6.
7. 8. 9. 10.
11.
12. 13.
14.
IS.
16.
Afcumulation of di-2-ethylhexyl phthalate (DEHP) in whole blood, platelet concentrates, and platelet-poor plasma. Transfusion 1434, 1974. Darby, T. D., and R. F. Wallin: Some quantitative aspects of toxicity. Pharmacologist 18171, 1976. Dixon, W. J.: BMDP Biomedical Computer Programs, (BMDPZV). Berkeley, University of California Press, 1975. Dunn, 0. J.: Multiple comparisons among means. J. Am. Stat. Assn. 5652, 1961. Fishman, W.H.: Plasma Enzymes. In:The Plasma Roteins, vol. 2. F. W. Putnam, Ed. New York, Academic Press, 1960, p 70. Horowitz, D. L., and L. D. Hoover: Nonlinear parameter estimation. 11. Analysis of Biomedical Data by Time Sharing. I. Nonlinear Regression Analysis, Naval Medical Research Institute Report No. 25, Project No. MR005.20-0287, 26 February 1970; modified by C. Peck and B. Barrett, Letterman Army Institute of Research, Presidio of San Francisco, CA, 1976. Lake, B. G., S. D. Gangolli, P. Grasso, and A. G. Lloyd: Studies on the hepatic effects of orally administered di-(2-ethylhexyl) phthalate in the rat. Toxicol. Appl. Pharmacol. 32355, 1975. Miller, W. V., Ed.: Technical Methods and Procedures of the American Association of Blood Banks, 6th ed. Washington, D.C., 1974. Miripol, J. E., andI. J. Stem: Decreasedaccumulation of phthalate plasticizer during storage of blood as packed cells. Transfusion 17:71. 1977. , P. J. Garvin, and R. F. Wallin: Contract NOI-HB-22990 Final Technical Progress Report: Role of lipids, plasma protein concentration and plasma lipoprotein concentration on DEHP accumulation in plasma. Travenol Laboratories, Morton Grove, Ill., September 1975, pp. 4. Peck, C. C.. and P. W. Albro: Letterman Army Institute of Research Notebook No. 5-302.1, Letterman Army Institute of Research, Presidio of San Francisco. CA, Nov. 10, 1976. page 95. ,and T. F. Zuck: Disposition and metabolism of DEHP in primates. Workshop on Adenine and Red Cell Preservation. In: Transcript of
-
-
Transfusion MWh-AMI 1579
Proceedings: Dept. Health, Education and Welfare, Food and Drug Administration, Bureau of Biologics, Washington, D.C., 1 October 1976. 17. Peterson, R. V.: Toxicology of plastic devices having contact with blood. Final Report, N.I.H. Contract NIH-NHLI-73-2908-B:35-41, 1975. 18. The Pharmacopeiaof the United States of America, 18th Revision, Anticoagulant Citrate Phosphate Dextrose Solution. Easton, PA, Mack Printing Co., 1970, p. 47. 19. Rubin, R. T.: DEHP leaching and toxicity. Workshop on Adenine and Red Cell Reservation. Transcript of Proceedings: Dept. Health, Education and Welfare, Food and Drug Administration, Bureau of Biologics, Washington, D.C. 20. Suguira, M..and M. Isobe: Studies on the mechanism of lipase reaction. 111. Adsorption of chromobacterium lipase on hydrophobic glass beads. Chem. Pharm. Bull. 24.72, 1976. 21. Swisher, S. N.: The introduction of adenine fortified blood preservatives: Introduction and an interpretation of its history. Transfusion 17:309, 1977. 22. Winer, B. J.: Statistical Principles in Experimental Design. New York, McGraw-Hill, 1962, p. 518. p. 518.
Carl C. Peck, M.D.. LTC. MC, Division of Blood Research, Letterman Army Institute of Research, Presidio of San Francisco. Daniel G. Odom, Ph.D., Cornell College, Mount Vernon, Iowa. Harold I. Friedman, M.D., Ph.D.. Department of Surgery, Health Science Center, University of Arizona. Tucson, Arizona. Phillip W. Albro, Ph.D., National Institutes of Environmental Health Sciences, Research Triangle Park, North Carolina. J. Ronald Hass, Ph.D., National Institutes of Environmental Health Sciences. John T. Brady. University of Colorado Medical Center, Denver, Colorado. SPS Don A. Jess, B.S.. Physical Sciences Assistant, Division of Blood Research, Letterman Army Institute of Research, Presidio of San Francisco.
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