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A new assay for β-thromboglobulin in urine
THROMBOSIS RESEARCH 64; 33-43,199l 0049-3848/91 $3.00 + .OO Printed in the USA. Copyright (c) 1991 Pergamon Press pk. All rights reserved.
A NEW ASSAY FOR BTHROMBGGLOBULIN
IN URINE
P. Hjemdahl, C. Pemeby, E. Theodorsson, N. Egberg and P.T. Larsson Department of Clinical Pharmacology, Department of Clinical Chemistry and Coagulation Laboratory, Karolinska Hospital, and Department of Pharmacology, Kamlinska Institute, S-104 01 Stockholm, Sweden (Received
8.3.1991;
accepted
in revised form 11.7.1991
by Editor B. Hessel)
ABSTRACT
Measurements of B-thromboglobulin (BTG) excretion in urine may be of value for “field” studies and due to problems with sampling artifacts for BTG in plasma. Previous studies have used a radioimmunoassay designed for plasma without characterizing the “BTG” immunoreactivity in urine. We describe modifications of the assay which increase its sensitivity and a sample work-up procedure using Sephadex G-25M columns separating high molecular weight (I-IMW) components (presumably intact l3TG) from low molecular weight (LMW) immunoreactivity (i.e. DTG fragments and/or non-specific interferences). The sensitivity of the assay (with 2.5 ml sample) is cl2 pg/ml HMW BTG. Inter- and intraassay coefficients of variation were 7-104. Only 33 (range 5-75)s of BTG immunoreactivity in urine represented HMW BTG. LMW immunoreactivity may be related to salt and other non-specific influences in the sample. Recoveries of BTG were quite variable (g-1001) in unextracted urines, but high and reproducible (80+2%) in the HMW fraction. Thus, nonspecific interferences with BTG measurements in certain urines are overcome by the separation step. Using Sephadex fractionation l3TG immunoreactivities in night urines (n=15) were: uH3 pg/ml in the HMW fraction, 70&8 pg/ml in the LMW fraction, and 85flO pg/ml by direct assay. HMW BTG increased in daytime samples (to 3of5 pg/ml; p
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contact with foreign surfaces (4, 13). Such problems might be overcome by urinary measurements if the OTG excreted into urine reflects 13TGlevels in plasma. Several authors have compared OTG determinations in plasma and urine, but found urinary measurements to be of limited value. Difficulties with detetminations in patients with renal disease (13) and/or urinary tract bleeding (9) have been pointed out, as have problems with regard to how to express results (pg/ml, @unit time or ng/mmol creatinine) (14). Reference values for DTG in urine have differed (7,9, 14, 15), suggesting methodological problems with the sensitivities and/or specificities of the assays. The analytical challenge is greater with urine, as only 4.5% of the concentration of BTG immunoreactivity in plasma, i.e. ~100 pg/ml, is found in urine (15). In the absence of sample clean-up procedures non-specific components might contribute to or interfere with the immunoreactivity determined in urine. It might also be expected that degradation products of l3TG are excreted into urine. In the present report we describe some characteristics of l3TG related material excreted in urine. A simple procedure for the fractionation of gTG related material in urine is suggested as pretreatment before measurements. Furthermore, some modifications needed to increase the sensitivity of the radioimmunoassay for OTG are suggested. MATERIALS Commercially available kits (IM-88) for OTG in plasma were purchased from the Radiochemical Centre (Amersham, UK). Standards supplied with the kits were used for recovery experiments. Donkey anti-rabbit antibody coated cellulose particles (Sac-Cel, IDS, Usworth Hall, WA) were used to separate bound and free tracer in the radioimmunoassay. Materials used for urine fractionation included: prepacked 5 cm Sephadex G-25M columns (PD-10; Pharmacia Fine Chemicals, Uppsala, Sweden), Centriconmicrofilters (Amicon Div., Grace Co, Danvers, MA), Sep-Pak Cl8 cartridges (Waters Associates, Milford, MA) and prepacked 100 mg Bond-Elut ODS 2Olnn columns pore size 130A(Analytichem International, Harbor City, CA). Reagent grade chemicals were obtained from various sources. METHODS Sampling and sample treatment Urines were collected in plastic containers without any additions. Samples were collected separately for nights and days and portions were stored at -8ooC until analysis. Upon thawing the urines were centrifuged (10 min 1400 x g at 4oC) to remove particles before analysis. Radioimmunoassay form% The sensitivity of the commercially obtained radioimmunoassay was increased by modifying incubation procedures. To increase buffering capacity (urine pH may vary considerably) the normal incubation buffer was substituted by a 75 mmol/l barbital buffer pH 8.6 containing 0.2% bovine serum albumin and 0.01% Triton-X-100.2OOl.tl urine or standard were incubated with 2OOcl.1 antiserum (diluted 10 times) during 24 h at 4OC. Thereafter the tracer (also diluted 10 times) was added and the tubes incubated a further 24 h in the cold. Free and bound tracer were separated by the addition of 100~1 Sac-Cel. After 30 min at room temperature 2 ml isotonic saline was added, the tubes were centrifuged (10 mitt, 1500 x g in the cold) and the supernatant was removed by suction. Radioactivity in the pellet was counted during 5 min in an LKB Wallac 1272 Clinigamma gamma counter (Pharmacia Diagnostics Norden AB, Uppsala, Sweden). Standard curves (7.8-2000 pg/ml BTG) were made up in incubation buffer. Assay conditions were adjusted so that 3040% of the tracer was bound in the absence of BTG. The most important modifications of the above described radioimmunoassay for DTG as compared to the procedure recommended in the kit are: the assay buffer, the dilution of the antiserum, the extended incubations (kit instructions recommend only 1 h with both tracer and
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antiserum, in stead of 24 + 24 h) and the separation of free and bound tracer (by ammonium sulphate precipitation in kit instructions). Sample work-up with Sephadex columns Commercially available Sephadex G-25M (PD- 10) columns were used as described by the manufacturer. The columns were equilibrated with 25 ml 15 mmol/I barbital buffer pH 8.6 containing 0.04% bovine serum albumin (i.e. the incubation buffer for the radioimmunoassay diluted 5 times). 2.5 ml sample was applied to the column. Thereafter, “fraction 1” was eluted in 3.5 ml 11 mmol/l barbital buffer pH 8.6 containing 0.03% bovine serum albumin. “Fraction 2” was then eluted in 1 ml of the 15 mmol/l buffer and, finally, “fraction 3” was eluted in 5 ml of the 11 mmol/l buffer. Fractions 1 and 3 were taken to dryness in a vacuum centrifuge (Speedvac Concentrator, Savant Industries Inc., Farmingdale, N.Y.) and resuspended in 500 and 750 p.l distilled water, respectively, to yield a final barbital concentration of 75 mmol/l a pH of 8.6. This increased the sensitivity of the assay, as fraction 1 was 5 x concentrated compared to the sample, and fraction 3 was 3.3 x concentrated. Alternative sample work-up procedures tested Ultrafiltration: 2 ml urine was centrifuged (45 min at 3800 x g; fixed angle rotor) through
Centriconfilters (MW cut-off 10 000). The ultrafiltrate was removed and its volume determined. The filters were then turned upside-down and respun (2 min 1000 x g) to collect the supematant, the volume of which was also determined. Both fractions were analyzed. Microcolumn chromatography: For Sep-Pak extraction the columns were preconditioned with 5 ml 0.06 M saline containing 0.1% uiflouroacetic acid and 1 mg/ml Polypep followed by 10 ml 0.06 M saline containing 80% methanol and 5 ml saline. 4 ml sample was added, after which the columns were rinsed with 2 ml saline with 40% methanol containing trifluoroacetic acid. BTG was then eluted in 4 ml 80% methanol containing triflouroacetic acid.
For Bond-Elut ODS 20 extraction the columns were preconditioned with water and 80% methanol, both containing 0.1% triflouroacetic acid and 0.1% triethyl amine. 4 ml urine was applied to the column. After washing (5 ml water containing 40% methanol, 0.1% triflouroacetic acid and 0.1% triethyl amine), BTG was eluted in 3 ml 80% methanol containing triflouroacetic acid and triethyl amine. Vacuum was applied to yield a flow rate of 5 rnl/min through the columns, which was found to be optimal. Methanol concentrations needed to elute OTG-like immunoreactivity were established by gradients containing O-100% methanol. Various additives (acetonitrile, ortho-phosphoric acid, EDTA, oxalic acid and acetic acid) were tested, as were various concentrations of triflouroacetic acid and triethyl amine, before establishing the above-mentioned conditions. Creatinine was measured by the Jaffe reaction, using a Monarch 2000 automated analyzer (Instrumentation Laboratories, IL Test TM18161560). Presence of hemoglobin was checked using BM test Ecur 4 (Boehringer Mannheim Gmbh). Methods and materials used inwther
attempts to characterizeJTG
in urine:
BTG was isolated according to Holt et al (16) from platelets harvested from 240 ml of platelet rich plasma. Rabbit antiserum against human DTG was obtained by repeated subcutaneous injections of isolated platelet KIG mixed with Freund’s complete adjuvans. The rabbits were bled four times at two week intervals and the serum collected. The IgG fraction was isolated from serum by ion exchange chromatography on DEAE Sephadex (Pharmacia). The IgG fraction was coupled to CNBr-Sepharose (Pharmacia) according to standard procedures. Isolation of urinary l3TG was performed on pooled urines from postoperative patients, which had been tested for the absence of hemoglobin. The crude centrifuged urine (600 ml) was applied to the anti-B‘IG column. After washing, the adsorbed material was eluted with 0.2 movl glycine buffer, pH 2.5. After elution the pH was adjusted to 8.5 with Tris base. The eluted material was concentrated by freeze drying and then applied to a preparative reverse-
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phase column (PepRPC HR 5/5, Pharmacia) developed with an acetonitrile-water gradient (2050%). Fractions containing BTG immunoreactive material were pooled and used for NH2terminal analyses in an Applied Biosynthesis 477A pulsed liquid sequencer. statistics:
Results are presented as mean values + SEM. Statistical analyses were performed using Statview II (Abacus Concepts, Inc., Berkely, CA) and a Macintosh IICi. Linear or non-linear regression analysis and Student’s paired or unpaired t-tests were used. RESULTS Pelfomance of modified radioimmunoassayfor$TG The above described modifications of the radioimmunoassay resulted in a lOO-fold increase in sensitivity, compared to that obtained when used according to instructions from the manufacturer (which were designed to yield rapid results for measurements in plasma). The linear portion of the standard curve (i.e. B/B0 = 80-20%) was =50-600 pg/ml. The detection limit of the assay (conservatively defined as the EDgo, i.e. B/B0 = 80%) varied with the batch of the kit, but was usually I60 (range 15-60) pg/ml with a sample volume of 200~1. Three of the immunized rabbits yielded high tine antibodies to standard OTG. When testing these fragments on HMW and LMW fractions from urine the results were similar to those obtained with the commercial antibodies. Thus, these new antibodies had similar binding characteristics as those supplied in the commercial kits; no analytical advantages were found. Sephaakx columnfractionation of standards Sephadex fractionation of standards (240 pg/ml) as described above yielded a recovery of 88s % (n=15) in “fraction l”, i.e. the HMW fraction. No detectable activity was found in “fraction 2” and 13fl % of the activity was determined in “fraction 3”, i.e. the LMW fraction. The latter activity, however, represented only approximately =20 pg/tube in the assay and is therefore uncertain. Direct andfractionated analysesofJTG immunoreactivityin urine Direct analysis of urines from 15 healthy volunteers (night samples) by the modified radioimmunoassay yielded “OTG” concentrations of 8213 pg/ml or 5.Olti.32 ng/mmol creatinine. The corresponding daytime values did not differ from these values and were 85flO pg/ml and 5.31Xl.44 ng/mmol creatinine, respectively (Fig. 1). Sephadex fractionation of these samples yielded 20.2k3.1 pg./ml HMW and 69.7k7.8 pg/ml LMW material (Fig. 1). Corresponding values corrected for creatinine were 1.35fo.25 and 4.67ti.55 ng/mmol creatinine, respectively. 26&3% of the immunoreactivity determined by direct assay was recovered in the HMW fraction and 97f12% in the LMW fraction of the night urines. For day urines the corresponding figures were 39&3 and lOlf14%, respectively. Immunoreactivity in “fraction 2” was at or below the detection limit. It is interesting to note that diurnal variability of urinary OTG-immunoreactivity was found only in the HMW fraction. Assay reproducibility Reproducibility of the assay, including the Sephadex fractionation step described above, was tested in samples containing normal concentrations of immunoreactivity. For the HMW fraction the coefficients of variation were 10.5% (intra-assay) and 7.0% (inter-assay). Similar assay variabilities were found for the LMW fraction and the direct assay. Recovery ofprC in urine Recoveries of BTG (240 pg/ml) added to urines were extremely variable when measured by direct assay. However, Fig. 2 illustrates that recoveries in the HMW fraction were similarly good (80+2 %) whether recovery by direct assay was poor or good in the individual sample. Omission of bovine serum albumin from the buffer reduced the recovery of BTG. Preconditioning of the Sephadex columns with Polypep (to reduce non-specific adsorption of peptide material) did not further enhance the recovery of BTG.
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fbTHROMBOGLOBULIN
DIRECT ASSAY -
IN URINE
37
SEPHADEX FRACTIONATION 1001
Low MW
100
High MW
1
80
1 60 40
1
I-
p < 0,Ol -I
-
65. 4. 3.
DAY
NIGHT
DAY
NIGHT
I- p
DAY
NIGHT
Fig. 1: Diurnal variationofflG immunereactivityin urine determined by direct assay and after separationintoHMW and LMW immunoreactivity. IncreasedJTG excretion was seen in daytimesamples only withregard to HMW material,whetherexpressed as the urinary concentrationper se (30 vs 20 pglml) or in relationto creatinineexcretion (2.02 vs I .35 nglmmol creatinine; p
o recovery by direct assay l recovery in HMW fraction
if? Y 8 Ls
O
0
‘l’l’l’l’l’l~i’l-l~l 2 4 6 8 10 12 14 16 18 20
Sample no. Fig. 2: Recoveries of JTG immunoreactivity by direct assay and in the HMW fraction (“fraction I “) after Sephadex fractionation in 20 samples. 240 pglmlJTG standard was added to each sample.
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Stability ofJTG in urine samples
l3TG immunoreactivity in urine was generally stable at room temperature. Occasionally, problems were encountered (Table 1). In samples with good recovery of DTG by direct assay HMW OTG was found to be stable 224 h at room temperature. In samples with poor recovery, however, there was a time dependent loss of HMW immunoreactivity, suggesting that analysis should be started within 1 h of thawing the samples. Repeated freezing and thawing of samples with poor recoveries resulted in further reductions of recoveries of spiked material. Loss of HMW activity was not accompanied by any build-up of LMW immunomactivity.
Goodrecoveries Immunoreactivity @g/ml)
Recovery (%)
HMW
LMW
79+21
49+16
2: 72
85f22 88+22 9Ok23
49f15 48f14 501k14
391t6 39+6 41f6 43+6
91+9 91+10 94k9 87k9
89k6
-
39+6
89&6 91f7 69k7
-
2326 14f4 3+1
Cl 2: 72
Poor recoveries Direct HMW LMW
Direct
102-4 92f18 92k20 105f17
25&7 81f15 22f6 8ti15 19k5 79k14 13f2 68+5 35+6 16+4 4f5 -
77f13 -
Table 1: JTG immunoreactivity over time (at room temperature) by direct assay and in the HMW and LMWfractions in 7 samples showing poor recovery of addedJTG (c 50% by direct assay) and in 7 samples in without recovery problems. Recovery values (%) for addedJTG (240 pglml) are also given.
Dilution of urine with good recovery of exogenous BTG by urines with poor recoveries reduced the recovery of BTG. Conversely, dilution of urine with poor recovery of BTG by incubation buffer increased the recovery of added BTG. We do not know which factor causes the concentration dependent interference with BTG measurements in some urines. The problem of poor recovery of BTG in unfiactionated urine was most common in night urines. Characterization of LMWJTG immunoreactivity and alternative extraction methods
There was a curvilinear relationship between osmolality and immunoreactivity in the LMW fraction (Fig. 3). Addition of NaCl to assay buffer without OTG created apparent BTG immune reactivity (60 pg/ml at 250 mM and by 105 pg/ml at 500 mM). Fig. 4 illustrates that addition of 300 mM NaCl to urine increased immunoreactivity from 56kl4 to 93f8 pg/ml by direct assay and from 5Ok6 to 71k6 pg/ml in the LMW fraction. Immunoreactivity in the HMW fraction, on the other hand, was not influenced. These results indicate that at least part of the “activity” in the LMW fraction represents nonspecific salt interferences with the assay. Reverse-phase extraction on Sep-Pak Cl8 columns resulted in unacceptably low recoveries (40%) of BTG both in buffer and in urine. Preconditioning of the columns with Polypep did not improve recoveries. The problem was caused by poor retention by these columns. Bond-Elut ODS 20~ columns were also tested, as their pore size (130A) should improve the retention of the large (36 000 MW; 4) BTG molecule. With appropriate preconditioning and elution (see Methods) the overall recovery of standards was 92k4 % (n=9). LMW immunoreactivity was not retained by the columns. Recovery of l3TG in urine was, however, quite variable (30-80%). Day urines generally showed a good recovery, but recoveries were low in some night urines, apparently due to tight binding to the columns. We failed to obtain dependably high recoveries of BTG with these columns despite efforts to ovemome the problems.
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C)-THROMBOGLOBULIN IN URINE
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y = 21.47 - 0.027x + 1.76 m1O-4x 2
p < 0.001
200
-
400
Osmolality
600
.
800
.
lb00
.
(mOsm/kg)
Fig. 3: Curvilinear relationship (pcO.001) between osmolality andjTG immunoreactivity in the LMWfraction in 24 randomly chosen urine samples.
ar(
l
?
pco.01
100
q no addition n 300mMNaCl
T pco.01 -I-
Direct assay
HMW fraction
LMW fraction
Fig.4: Influence of 300 mM NaCl onJTG immunoreactivity in urine by direct assay and ajier fractionation injive urines with sodium concentrations of 103-19.5 mM before additions.
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fbTHROMBOGLOBULIN
Vol. 64, No. 1
IN URINE
Ultrafiltration of l3TG standards using Centriconfilters resulted in recoveries of 85% above the filter (supematant) and 3% (i.e. barely measurable levels) in the ultrafiltrate. Similar fractionation of four urines (night samples) with 115k29 pg/ml DTG immunoreactivity by direct assay yielded values of 108+33 pg/ml in the ultrafiltrate and 29f8 pg/ml in the supematant. Calculation of HMW BTG concentrations were impaired by variable volumes of urine retained above the filter. Thus, the ultrafiltration method was not practical for the purpose of estimating HMW BTG. However, there was a linear correlation between immunoreactivity in fraction 3 from Sephadex columns and ultrafiltrates (Fig. 5), indicating that fraction 3 indeed represents LMW immunoreactivity present in urine before sample treatment. 180 . 160 3 E ;;a ,a 8 ‘3 3 a 9 2
140 120, 100 80 60, 40. 20 0’
20
40
60
80
100
120
140
160
Ultrafiltrate (pg/ml)
Fig. 5: Relationship betweenJTG immunoreactivity in ultrafiltrates (Centricon IO 000 MW cut-off filters) and in the LMW fraction obtained by Sephadex chromatography in I1 urine samples. There was a highly significant (pcO.001) correlation between results obtained with the two methoak of separation. Further characterization ofJTG immunoreactivity in urine Attempts to further characterize the KIG immunomactivity in urine by antibody-based extraction of urine followed by reverse-phase chromatography and NI+terminal analysis were partly inconclusive, but confvmed the presence of an inhomogenous LMW fraction of BTG immunoreactive material. A number of NI-IZ-terminal amino acids were identified. They were, mainly, Asp, Asn, Glu, Tyr, Met and Val, all of which could represent various fragments of the l3TG subunit. Further identification of the immunoreactive material was impossible. DISCUSSION The present method for selective determination of HMW BTG immunomactivity in urine has improved the possibilities of obtaining relevant information on BTG release from platelets in vivo in several ways: First, as a prerequisite for measurements of the low levels of HMW OTG immunoreactivity, we have described a way to improve the sensitivity of the assay. The present modifications of the assay can equally well be used for measurements of OTG in plasma. This modified assay is cheaper but more time consuming than the rapid assay described in the kit. The sensitivity of the assay can be increased to about 30 pg/ml. Measurements should preferably be performed in
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fbTHROMBOGLOBULIN
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the straight portion of the standard curve, which requites an approximate 2OO-fold dilution of plasma (levels in plasma are usually 20-50 ng/ml). Occasionally, HMW l3TG immunomactivity in urine will be near the detection limit. Second, we have shown that there are problems with poor recoveries of exogenous OTG in some samples, but that selective determination of HMW DTG virtually cirumvents them. Some so far uncharacterized substance(s) in the LMW fraction of urine seem to interfere with the radioimmunoassay for l3TG. Such interference with measurements of BTG may be reduced by dilution or removed by gel chromatography, which leaves only HMW material in the assay. Third, we have shown that the majority of OTG immunoreactivity in urine represents nonspecific activity. A simple factor such as the salt concentration (i.e. osmolality) of the sample influences values obtained with the direct assay and correlates with DTG immunoreactivity in the LMW fraction obtained after Sephadex fractionation or after ultrafiltration. We found no evidence for breakdown of intact DTG to immunoreactive fragments in urines displaying decay of activity over time in vifro, as loss of OTG activity in the HMW fraction was not accompanied by any build-up of activity in the LMW fraction. The large number of compounds with different NHZ-terminals found in our study suggests that there was a heterogenous mixture of peptides with remaining immunoreactivity. Thus, there is presumably breakdown of BTG to immunoreactive fragments before excretion into urine. Whether our HMW fraction represents only intact STG or several large and immunologically recognizable fragments can not be determined from the present results. Our results suggest that the previously determined fractional excretion of BTG into urine (0.5%; 15) has been over-estimated. Our data would imply a fractional excretion of about 0.1%. Fourth, we have shown that OTG immunoreactivity in the HMW fraction obtained by Sephadex chromatography displays the diurnal variability which is expected to be found with regard to platelet function (17). Diurnal variability was not found with measurements of LMW immunoreactivity (non-specific elements?) or when assaying unextracted urine directly. Since the LMW fraction contained most of the l3TG immunoreactivity in urine (67%, on the average, with a wide range of 2595%). it seems as if non-specific LMW components obscure true changes in OTG release and excretion. Direct assays may not reveal biological variation unless the variations studied are very large due to this non-specific background of LMW activity. One of the problems not circumvented by the presently improved assay for immunoreactive OTG in urine is urinary tract bleeding, as a small amount of blood in the sample may result in artifactual release of OTG from the platelets. Thus, samples should be checked for hemoglobin contents and discarded if such contents are found to be high (9). We have only checked this with simple dip slides. It will be of interest to perform quantitative measurements and to try to establish a suitable cut-off point for hemoglobin in urine. The present method is especially suitable for studies of l3TG release in vivo over longer time periods and in settings where blood sampling are impractical, such as ‘Yield” studies. However, it should be noted that plasma measurements of OTG immunoreactivity have draw backs also for short term studies, due to the well-known problems with sampling artifacts. In addition, the long half life of l3TG in plasma (= 100 min; 15) results in a need for long resting periods prior to basal sampling when studying BTG release during physiological interventions in vivo (18). With regard to the problem of how to express data (14), we found similar results whether expressed as concentrations per se or in relation to creatinine excretion (Fig. 1). The latter method, however, seems most attractive to us and eliminates the need for exact estimates of the time during which the urine was produced (expression per unit time) or for quantitative (and often unreliable) 24 h urine collections, which were the methods recommended by Musumeci er al (14). When expressing l3TG excretion per unit cm&nine these problems disappear. In summary, the present assay for selective determinations of HMW OTG in urine which is also based on an improved sensitivity of the radioimmunoassay, provides a much improved
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tool for studies of platelet release reactions in vivo. Our data suggest that physiologically meaningful changes (as exemplified by diurnal variation) may be picked up only when the modified assay is used. The value of selective measurements of HMW BTG immunoreactivity in urine will be determined by further physiological and pathophysiological studies. ACKNOWLEDGEMENTS The study was supported by grants from the Swedish Heart-Lung Foundation and the Swedish Medical Research Council (5930). We are also grateful for receiving partial supplies of materials used to establish sample work-up procedures, i.e. some of the PD-10 columns (Pharmacia, Uppsala, Sweden), some of the Bond-Elut ODS 2Opm columns (Sorbent AB, Vlstra Friilunda, Sweden) and some of the filters used for ultrafiltration from Grace Co. (Mahno, Sweden). G. Oresten of Sorbent AB made helpful suggestions with regard to reverse phase column extractions. The attempts of professor Hans Jomvall, Dept. of Biochemistry, Karolinska Institute, to sequence the BTG-related material in urine are gratefully acknowledged. The expert laboratory assistance of Ms. Caroline Netre is also gratefully acknowledged. REFERENCES 1. LUDLAM, C.A., MOORE, S. BOLTON, A.E., PEPPER, D.S. and- CASH, J.D. The release of a human platelet specific protein measured by a radioimmunoassay. ThrombosisRes. 6: 543-548, 1975. 2. MOORE, S., PEPPER, D.S. and CASH, J.D. The isolation and characterization of a platelet specific O-globulin (&thromboglobulin) and the detection of anti-urokinase and antiplasmin released from thrombin aggregated washed human platelets. Biochim. Biophys. Actu 379: 360-369, 1975. 3. KAPLAN, K.L., NOSSEL, H.L., DRILLINGS, M. and LESZNIK, G. Radioimmunoassay of platelet factor 4 and B-thromboglobulin. Development and application to studies on platelet release in relation to fibrinopeptide A generation. Br. J. Huemufol. 39: 129-146, 1978. 4. KAPLAN, K.L. and OWEN, J. Plasma levels of platelet secretory proteins. CRC Crir. Rev. Oncol. Hematol. 5: 235-255,1986 5. BEGG, G.S., PEPPER, D.S., CHESTERMAN, C.N. and MORGAN, F.J. Complete covalent structure of human l3-thromboglobulin. Biochemistry27: 1739-1744,1978. 6. BURROWS, A.W., CHAVIN, S.I. and HOCKADAY, T.D.R. thromboglobulin concentrations in diabetes mellitus. Luncet i: 235-237,1978.
Plasma
S-
7. VAN OOST, B.A., VELDHUYZEN, B. TIMMERMANS, A.P.M. and SIXMA, J.J. Increased urinary O-thromboglobulin excretion in diabetes assayed with a modified RIA kit technique. Thromb. Haemost. 49: 18-20, 1983. 8. CELLA, G., ZAHAVI, J., DE HASS, H.A. and KAKKAR, V.V. D-Thromboglobulin, platelet production time and platelet function in vascular disease. Br. J. Huemafol. 43: 127-136, 1979. 9. BOLTON, A.E., COOKE, E.D., LEKI-IWANI, C.P. and BOWCOCK, S.A. Urinary l3thromboglobulin as a diagnostic marker for post-operative deep vein thrombosis. Thrombosis Res. 19: 249-255, 1980. 10. DE BOER, A.C., TURPIE, A.G.G., BUTT, R., ZIELINSKY, A. and GENTON, E. Plasma and urine D-thromboglobulin concentration in patients with deep vein thrombosis. Blood 58: 693-698, 1981.
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11. DOYLE, D.J., CHESTERMAN, C.N., CADE, J.F. MeGREADY, J.R., RENNIE, G.C. and MORGAN, F.J. Plasma concentrations of platelet specific proteins correlated with platelet survival. Blood 55: 82-84, 1980. 12. BOLTON, A.E., LUDLAM, C.A., MOORE, S. PEPPER, D.S. and CASH, J.D. Three approaches to the radioimmunoassay of human B-thromboglobulin. Br. J. Haemarol. 33: 233239, 1976. 13. HOPPER, A.H., TINDALL, M. and DAVIES, J.A. Urinary l3-thromboglobulin correlates with impairment of renal function in patients with diabetic nephropathy. Thromb. Haemost. 56: 229-231, 1986.
14. MUSUMECI, V., ROSA, S., CARUSO, A., ZUPPI, C. Urine B-thromboglobulin concentration ratio in single voided urine samples cannot be thromboglobulin excretion. Thromb. Haemost. 55:
ZAPPACOSTA, B., TUTINELLI, F. and or l3-thromboglobulin/creatinine excretion reliably used to estimate quantitative B2-5, 1986.
15. DAWES, J., SMITH, R.C. and PEPPER, D.S. The release, distribution, and clearance of human &thromboglobulin and platelet factor 4. ThrombosisRes. 12: 851-861, 1978. 16. HOLT, J.C., HARRIS, M.E., HOLT, A.M., LANGE, E., HENSCHEN, A. and NIEWAROWSKI, S. Characterization of human platelet basic protein, a precursor form of low-affinity platelet factor 4 and 8-thromboglobulin. Biochemistry25: 1988-1996, 1986. 17. TOFLER, G.H., BREZINSKI, D., SCHAFER, A.I., CZEISLER, C.A., RUTHERFORD, J.D., WILLICH, S.N., GLEASON, R.E., WILLIAMS, G.H. and MULLER, J.E. Concurrent morning increase in platelet aggregability and the risk for myocardial infarction and sudden cardiac death. New Engl. J. Med. 316: 1514-1518,1987. 18. LARSSON, P.T., HJEMDAHL, P., OLSSON, G., EGBERG, N. and HORNSTRA, G. Altered platelet function during mental stress and adrenaline infusions in humans: evidence for an increased aggregability in vivo as measured by filtragometry. Clin. Sci. 76: 369-376,1989.
43
A NEW ASSAY FOR BTHROMBGGLOBULIN
IN URINE
P. Hjemdahl, C. Pemeby, E. Theodorsson, N. Egberg and P.T. Larsson Department of Clinical Pharmacology, Department of Clinical Chemistry and Coagulation Laboratory, Karolinska Hospital, and Department of Pharmacology, Kamlinska Institute, S-104 01 Stockholm, Sweden (Received
8.3.1991;
accepted
in revised form 11.7.1991
by Editor B. Hessel)
ABSTRACT
Measurements of B-thromboglobulin (BTG) excretion in urine may be of value for “field” studies and due to problems with sampling artifacts for BTG in plasma. Previous studies have used a radioimmunoassay designed for plasma without characterizing the “BTG” immunoreactivity in urine. We describe modifications of the assay which increase its sensitivity and a sample work-up procedure using Sephadex G-25M columns separating high molecular weight (I-IMW) components (presumably intact l3TG) from low molecular weight (LMW) immunoreactivity (i.e. DTG fragments and/or non-specific interferences). The sensitivity of the assay (with 2.5 ml sample) is cl2 pg/ml HMW BTG. Inter- and intraassay coefficients of variation were 7-104. Only 33 (range 5-75)s of BTG immunoreactivity in urine represented HMW BTG. LMW immunoreactivity may be related to salt and other non-specific influences in the sample. Recoveries of BTG were quite variable (g-1001) in unextracted urines, but high and reproducible (80+2%) in the HMW fraction. Thus, nonspecific interferences with BTG measurements in certain urines are overcome by the separation step. Using Sephadex fractionation l3TG immunoreactivities in night urines (n=15) were: uH3 pg/ml in the HMW fraction, 70&8 pg/ml in the LMW fraction, and 85flO pg/ml by direct assay. HMW BTG increased in daytime samples (to 3of5 pg/ml; p
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contact with foreign surfaces (4, 13). Such problems might be overcome by urinary measurements if the OTG excreted into urine reflects 13TGlevels in plasma. Several authors have compared OTG determinations in plasma and urine, but found urinary measurements to be of limited value. Difficulties with detetminations in patients with renal disease (13) and/or urinary tract bleeding (9) have been pointed out, as have problems with regard to how to express results (pg/ml, @unit time or ng/mmol creatinine) (14). Reference values for DTG in urine have differed (7,9, 14, 15), suggesting methodological problems with the sensitivities and/or specificities of the assays. The analytical challenge is greater with urine, as only 4.5% of the concentration of BTG immunoreactivity in plasma, i.e. ~100 pg/ml, is found in urine (15). In the absence of sample clean-up procedures non-specific components might contribute to or interfere with the immunoreactivity determined in urine. It might also be expected that degradation products of l3TG are excreted into urine. In the present report we describe some characteristics of l3TG related material excreted in urine. A simple procedure for the fractionation of gTG related material in urine is suggested as pretreatment before measurements. Furthermore, some modifications needed to increase the sensitivity of the radioimmunoassay for OTG are suggested. MATERIALS Commercially available kits (IM-88) for OTG in plasma were purchased from the Radiochemical Centre (Amersham, UK). Standards supplied with the kits were used for recovery experiments. Donkey anti-rabbit antibody coated cellulose particles (Sac-Cel, IDS, Usworth Hall, WA) were used to separate bound and free tracer in the radioimmunoassay. Materials used for urine fractionation included: prepacked 5 cm Sephadex G-25M columns (PD-10; Pharmacia Fine Chemicals, Uppsala, Sweden), Centriconmicrofilters (Amicon Div., Grace Co, Danvers, MA), Sep-Pak Cl8 cartridges (Waters Associates, Milford, MA) and prepacked 100 mg Bond-Elut ODS 2Olnn columns pore size 130A(Analytichem International, Harbor City, CA). Reagent grade chemicals were obtained from various sources. METHODS Sampling and sample treatment Urines were collected in plastic containers without any additions. Samples were collected separately for nights and days and portions were stored at -8ooC until analysis. Upon thawing the urines were centrifuged (10 min 1400 x g at 4oC) to remove particles before analysis. Radioimmunoassay form% The sensitivity of the commercially obtained radioimmunoassay was increased by modifying incubation procedures. To increase buffering capacity (urine pH may vary considerably) the normal incubation buffer was substituted by a 75 mmol/l barbital buffer pH 8.6 containing 0.2% bovine serum albumin and 0.01% Triton-X-100.2OOl.tl urine or standard were incubated with 2OOcl.1 antiserum (diluted 10 times) during 24 h at 4OC. Thereafter the tracer (also diluted 10 times) was added and the tubes incubated a further 24 h in the cold. Free and bound tracer were separated by the addition of 100~1 Sac-Cel. After 30 min at room temperature 2 ml isotonic saline was added, the tubes were centrifuged (10 mitt, 1500 x g in the cold) and the supernatant was removed by suction. Radioactivity in the pellet was counted during 5 min in an LKB Wallac 1272 Clinigamma gamma counter (Pharmacia Diagnostics Norden AB, Uppsala, Sweden). Standard curves (7.8-2000 pg/ml BTG) were made up in incubation buffer. Assay conditions were adjusted so that 3040% of the tracer was bound in the absence of BTG. The most important modifications of the above described radioimmunoassay for DTG as compared to the procedure recommended in the kit are: the assay buffer, the dilution of the antiserum, the extended incubations (kit instructions recommend only 1 h with both tracer and
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antiserum, in stead of 24 + 24 h) and the separation of free and bound tracer (by ammonium sulphate precipitation in kit instructions). Sample work-up with Sephadex columns Commercially available Sephadex G-25M (PD- 10) columns were used as described by the manufacturer. The columns were equilibrated with 25 ml 15 mmol/I barbital buffer pH 8.6 containing 0.04% bovine serum albumin (i.e. the incubation buffer for the radioimmunoassay diluted 5 times). 2.5 ml sample was applied to the column. Thereafter, “fraction 1” was eluted in 3.5 ml 11 mmol/l barbital buffer pH 8.6 containing 0.03% bovine serum albumin. “Fraction 2” was then eluted in 1 ml of the 15 mmol/l buffer and, finally, “fraction 3” was eluted in 5 ml of the 11 mmol/l buffer. Fractions 1 and 3 were taken to dryness in a vacuum centrifuge (Speedvac Concentrator, Savant Industries Inc., Farmingdale, N.Y.) and resuspended in 500 and 750 p.l distilled water, respectively, to yield a final barbital concentration of 75 mmol/l a pH of 8.6. This increased the sensitivity of the assay, as fraction 1 was 5 x concentrated compared to the sample, and fraction 3 was 3.3 x concentrated. Alternative sample work-up procedures tested Ultrafiltration: 2 ml urine was centrifuged (45 min at 3800 x g; fixed angle rotor) through
Centriconfilters (MW cut-off 10 000). The ultrafiltrate was removed and its volume determined. The filters were then turned upside-down and respun (2 min 1000 x g) to collect the supematant, the volume of which was also determined. Both fractions were analyzed. Microcolumn chromatography: For Sep-Pak extraction the columns were preconditioned with 5 ml 0.06 M saline containing 0.1% uiflouroacetic acid and 1 mg/ml Polypep followed by 10 ml 0.06 M saline containing 80% methanol and 5 ml saline. 4 ml sample was added, after which the columns were rinsed with 2 ml saline with 40% methanol containing trifluoroacetic acid. BTG was then eluted in 4 ml 80% methanol containing triflouroacetic acid.
For Bond-Elut ODS 20 extraction the columns were preconditioned with water and 80% methanol, both containing 0.1% triflouroacetic acid and 0.1% triethyl amine. 4 ml urine was applied to the column. After washing (5 ml water containing 40% methanol, 0.1% triflouroacetic acid and 0.1% triethyl amine), BTG was eluted in 3 ml 80% methanol containing triflouroacetic acid and triethyl amine. Vacuum was applied to yield a flow rate of 5 rnl/min through the columns, which was found to be optimal. Methanol concentrations needed to elute OTG-like immunoreactivity were established by gradients containing O-100% methanol. Various additives (acetonitrile, ortho-phosphoric acid, EDTA, oxalic acid and acetic acid) were tested, as were various concentrations of triflouroacetic acid and triethyl amine, before establishing the above-mentioned conditions. Creatinine was measured by the Jaffe reaction, using a Monarch 2000 automated analyzer (Instrumentation Laboratories, IL Test TM18161560). Presence of hemoglobin was checked using BM test Ecur 4 (Boehringer Mannheim Gmbh). Methods and materials used inwther
attempts to characterizeJTG
in urine:
BTG was isolated according to Holt et al (16) from platelets harvested from 240 ml of platelet rich plasma. Rabbit antiserum against human DTG was obtained by repeated subcutaneous injections of isolated platelet KIG mixed with Freund’s complete adjuvans. The rabbits were bled four times at two week intervals and the serum collected. The IgG fraction was isolated from serum by ion exchange chromatography on DEAE Sephadex (Pharmacia). The IgG fraction was coupled to CNBr-Sepharose (Pharmacia) according to standard procedures. Isolation of urinary l3TG was performed on pooled urines from postoperative patients, which had been tested for the absence of hemoglobin. The crude centrifuged urine (600 ml) was applied to the anti-B‘IG column. After washing, the adsorbed material was eluted with 0.2 movl glycine buffer, pH 2.5. After elution the pH was adjusted to 8.5 with Tris base. The eluted material was concentrated by freeze drying and then applied to a preparative reverse-
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phase column (PepRPC HR 5/5, Pharmacia) developed with an acetonitrile-water gradient (2050%). Fractions containing BTG immunoreactive material were pooled and used for NH2terminal analyses in an Applied Biosynthesis 477A pulsed liquid sequencer. statistics:
Results are presented as mean values + SEM. Statistical analyses were performed using Statview II (Abacus Concepts, Inc., Berkely, CA) and a Macintosh IICi. Linear or non-linear regression analysis and Student’s paired or unpaired t-tests were used. RESULTS Pelfomance of modified radioimmunoassayfor$TG The above described modifications of the radioimmunoassay resulted in a lOO-fold increase in sensitivity, compared to that obtained when used according to instructions from the manufacturer (which were designed to yield rapid results for measurements in plasma). The linear portion of the standard curve (i.e. B/B0 = 80-20%) was =50-600 pg/ml. The detection limit of the assay (conservatively defined as the EDgo, i.e. B/B0 = 80%) varied with the batch of the kit, but was usually I60 (range 15-60) pg/ml with a sample volume of 200~1. Three of the immunized rabbits yielded high tine antibodies to standard OTG. When testing these fragments on HMW and LMW fractions from urine the results were similar to those obtained with the commercial antibodies. Thus, these new antibodies had similar binding characteristics as those supplied in the commercial kits; no analytical advantages were found. Sephaakx columnfractionation of standards Sephadex fractionation of standards (240 pg/ml) as described above yielded a recovery of 88s % (n=15) in “fraction l”, i.e. the HMW fraction. No detectable activity was found in “fraction 2” and 13fl % of the activity was determined in “fraction 3”, i.e. the LMW fraction. The latter activity, however, represented only approximately =20 pg/tube in the assay and is therefore uncertain. Direct andfractionated analysesofJTG immunoreactivityin urine Direct analysis of urines from 15 healthy volunteers (night samples) by the modified radioimmunoassay yielded “OTG” concentrations of 8213 pg/ml or 5.Olti.32 ng/mmol creatinine. The corresponding daytime values did not differ from these values and were 85flO pg/ml and 5.31Xl.44 ng/mmol creatinine, respectively (Fig. 1). Sephadex fractionation of these samples yielded 20.2k3.1 pg./ml HMW and 69.7k7.8 pg/ml LMW material (Fig. 1). Corresponding values corrected for creatinine were 1.35fo.25 and 4.67ti.55 ng/mmol creatinine, respectively. 26&3% of the immunoreactivity determined by direct assay was recovered in the HMW fraction and 97f12% in the LMW fraction of the night urines. For day urines the corresponding figures were 39&3 and lOlf14%, respectively. Immunoreactivity in “fraction 2” was at or below the detection limit. It is interesting to note that diurnal variability of urinary OTG-immunoreactivity was found only in the HMW fraction. Assay reproducibility Reproducibility of the assay, including the Sephadex fractionation step described above, was tested in samples containing normal concentrations of immunoreactivity. For the HMW fraction the coefficients of variation were 10.5% (intra-assay) and 7.0% (inter-assay). Similar assay variabilities were found for the LMW fraction and the direct assay. Recovery ofprC in urine Recoveries of BTG (240 pg/ml) added to urines were extremely variable when measured by direct assay. However, Fig. 2 illustrates that recoveries in the HMW fraction were similarly good (80+2 %) whether recovery by direct assay was poor or good in the individual sample. Omission of bovine serum albumin from the buffer reduced the recovery of BTG. Preconditioning of the Sephadex columns with Polypep (to reduce non-specific adsorption of peptide material) did not further enhance the recovery of BTG.
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37
SEPHADEX FRACTIONATION 1001
Low MW
100
High MW
1
80
1 60 40
1
I-
p < 0,Ol -I
-
65. 4. 3.
DAY
NIGHT
DAY
NIGHT
I- p
DAY
NIGHT
Fig. 1: Diurnal variationofflG immunereactivityin urine determined by direct assay and after separationintoHMW and LMW immunoreactivity. IncreasedJTG excretion was seen in daytimesamples only withregard to HMW material,whetherexpressed as the urinary concentrationper se (30 vs 20 pglml) or in relationto creatinineexcretion (2.02 vs I .35 nglmmol creatinine; p
o recovery by direct assay l recovery in HMW fraction
if? Y 8 Ls
O
0
‘l’l’l’l’l’l~i’l-l~l 2 4 6 8 10 12 14 16 18 20
Sample no. Fig. 2: Recoveries of JTG immunoreactivity by direct assay and in the HMW fraction (“fraction I “) after Sephadex fractionation in 20 samples. 240 pglmlJTG standard was added to each sample.
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Stability ofJTG in urine samples
l3TG immunoreactivity in urine was generally stable at room temperature. Occasionally, problems were encountered (Table 1). In samples with good recovery of DTG by direct assay HMW OTG was found to be stable 224 h at room temperature. In samples with poor recovery, however, there was a time dependent loss of HMW immunoreactivity, suggesting that analysis should be started within 1 h of thawing the samples. Repeated freezing and thawing of samples with poor recoveries resulted in further reductions of recoveries of spiked material. Loss of HMW activity was not accompanied by any build-up of LMW immunomactivity.
Goodrecoveries Immunoreactivity @g/ml)
Recovery (%)
HMW
LMW
79+21
49+16
2: 72
85f22 88+22 9Ok23
49f15 48f14 501k14
391t6 39+6 41f6 43+6
91+9 91+10 94k9 87k9
89k6
-
39+6
89&6 91f7 69k7
-
2326 14f4 3+1
Cl 2: 72
Poor recoveries Direct HMW LMW
Direct
102-4 92f18 92k20 105f17
25&7 81f15 22f6 8ti15 19k5 79k14 13f2 68+5 35+6 16+4 4f5 -
77f13 -
Table 1: JTG immunoreactivity over time (at room temperature) by direct assay and in the HMW and LMWfractions in 7 samples showing poor recovery of addedJTG (c 50% by direct assay) and in 7 samples in without recovery problems. Recovery values (%) for addedJTG (240 pglml) are also given.
Dilution of urine with good recovery of exogenous BTG by urines with poor recoveries reduced the recovery of BTG. Conversely, dilution of urine with poor recovery of BTG by incubation buffer increased the recovery of added BTG. We do not know which factor causes the concentration dependent interference with BTG measurements in some urines. The problem of poor recovery of BTG in unfiactionated urine was most common in night urines. Characterization of LMWJTG immunoreactivity and alternative extraction methods
There was a curvilinear relationship between osmolality and immunoreactivity in the LMW fraction (Fig. 3). Addition of NaCl to assay buffer without OTG created apparent BTG immune reactivity (60 pg/ml at 250 mM and by 105 pg/ml at 500 mM). Fig. 4 illustrates that addition of 300 mM NaCl to urine increased immunoreactivity from 56kl4 to 93f8 pg/ml by direct assay and from 5Ok6 to 71k6 pg/ml in the LMW fraction. Immunoreactivity in the HMW fraction, on the other hand, was not influenced. These results indicate that at least part of the “activity” in the LMW fraction represents nonspecific salt interferences with the assay. Reverse-phase extraction on Sep-Pak Cl8 columns resulted in unacceptably low recoveries (40%) of BTG both in buffer and in urine. Preconditioning of the columns with Polypep did not improve recoveries. The problem was caused by poor retention by these columns. Bond-Elut ODS 20~ columns were also tested, as their pore size (130A) should improve the retention of the large (36 000 MW; 4) BTG molecule. With appropriate preconditioning and elution (see Methods) the overall recovery of standards was 92k4 % (n=9). LMW immunoreactivity was not retained by the columns. Recovery of l3TG in urine was, however, quite variable (30-80%). Day urines generally showed a good recovery, but recoveries were low in some night urines, apparently due to tight binding to the columns. We failed to obtain dependably high recoveries of BTG with these columns despite efforts to ovemome the problems.
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y = 21.47 - 0.027x + 1.76 m1O-4x 2
p < 0.001
200
-
400
Osmolality
600
.
800
.
lb00
.
(mOsm/kg)
Fig. 3: Curvilinear relationship (pcO.001) between osmolality andjTG immunoreactivity in the LMWfraction in 24 randomly chosen urine samples.
ar(
l
?
pco.01
100
q no addition n 300mMNaCl
T pco.01 -I-
Direct assay
HMW fraction
LMW fraction
Fig.4: Influence of 300 mM NaCl onJTG immunoreactivity in urine by direct assay and ajier fractionation injive urines with sodium concentrations of 103-19.5 mM before additions.
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Ultrafiltration of l3TG standards using Centriconfilters resulted in recoveries of 85% above the filter (supematant) and 3% (i.e. barely measurable levels) in the ultrafiltrate. Similar fractionation of four urines (night samples) with 115k29 pg/ml DTG immunoreactivity by direct assay yielded values of 108+33 pg/ml in the ultrafiltrate and 29f8 pg/ml in the supematant. Calculation of HMW BTG concentrations were impaired by variable volumes of urine retained above the filter. Thus, the ultrafiltration method was not practical for the purpose of estimating HMW BTG. However, there was a linear correlation between immunoreactivity in fraction 3 from Sephadex columns and ultrafiltrates (Fig. 5), indicating that fraction 3 indeed represents LMW immunoreactivity present in urine before sample treatment. 180 . 160 3 E ;;a ,a 8 ‘3 3 a 9 2
140 120, 100 80 60, 40. 20 0’
20
40
60
80
100
120
140
160
Ultrafiltrate (pg/ml)
Fig. 5: Relationship betweenJTG immunoreactivity in ultrafiltrates (Centricon IO 000 MW cut-off filters) and in the LMW fraction obtained by Sephadex chromatography in I1 urine samples. There was a highly significant (pcO.001) correlation between results obtained with the two methoak of separation. Further characterization ofJTG immunoreactivity in urine Attempts to further characterize the KIG immunomactivity in urine by antibody-based extraction of urine followed by reverse-phase chromatography and NI+terminal analysis were partly inconclusive, but confvmed the presence of an inhomogenous LMW fraction of BTG immunoreactive material. A number of NI-IZ-terminal amino acids were identified. They were, mainly, Asp, Asn, Glu, Tyr, Met and Val, all of which could represent various fragments of the l3TG subunit. Further identification of the immunoreactive material was impossible. DISCUSSION The present method for selective determination of HMW BTG immunomactivity in urine has improved the possibilities of obtaining relevant information on BTG release from platelets in vivo in several ways: First, as a prerequisite for measurements of the low levels of HMW OTG immunoreactivity, we have described a way to improve the sensitivity of the assay. The present modifications of the assay can equally well be used for measurements of OTG in plasma. This modified assay is cheaper but more time consuming than the rapid assay described in the kit. The sensitivity of the assay can be increased to about 30 pg/ml. Measurements should preferably be performed in
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the straight portion of the standard curve, which requites an approximate 2OO-fold dilution of plasma (levels in plasma are usually 20-50 ng/ml). Occasionally, HMW l3TG immunomactivity in urine will be near the detection limit. Second, we have shown that there are problems with poor recoveries of exogenous OTG in some samples, but that selective determination of HMW DTG virtually cirumvents them. Some so far uncharacterized substance(s) in the LMW fraction of urine seem to interfere with the radioimmunoassay for l3TG. Such interference with measurements of BTG may be reduced by dilution or removed by gel chromatography, which leaves only HMW material in the assay. Third, we have shown that the majority of OTG immunoreactivity in urine represents nonspecific activity. A simple factor such as the salt concentration (i.e. osmolality) of the sample influences values obtained with the direct assay and correlates with DTG immunoreactivity in the LMW fraction obtained after Sephadex fractionation or after ultrafiltration. We found no evidence for breakdown of intact DTG to immunoreactive fragments in urines displaying decay of activity over time in vifro, as loss of OTG activity in the HMW fraction was not accompanied by any build-up of activity in the LMW fraction. The large number of compounds with different NHZ-terminals found in our study suggests that there was a heterogenous mixture of peptides with remaining immunoreactivity. Thus, there is presumably breakdown of BTG to immunoreactive fragments before excretion into urine. Whether our HMW fraction represents only intact STG or several large and immunologically recognizable fragments can not be determined from the present results. Our results suggest that the previously determined fractional excretion of BTG into urine (0.5%; 15) has been over-estimated. Our data would imply a fractional excretion of about 0.1%. Fourth, we have shown that OTG immunoreactivity in the HMW fraction obtained by Sephadex chromatography displays the diurnal variability which is expected to be found with regard to platelet function (17). Diurnal variability was not found with measurements of LMW immunoreactivity (non-specific elements?) or when assaying unextracted urine directly. Since the LMW fraction contained most of the l3TG immunoreactivity in urine (67%, on the average, with a wide range of 2595%). it seems as if non-specific LMW components obscure true changes in OTG release and excretion. Direct assays may not reveal biological variation unless the variations studied are very large due to this non-specific background of LMW activity. One of the problems not circumvented by the presently improved assay for immunoreactive OTG in urine is urinary tract bleeding, as a small amount of blood in the sample may result in artifactual release of OTG from the platelets. Thus, samples should be checked for hemoglobin contents and discarded if such contents are found to be high (9). We have only checked this with simple dip slides. It will be of interest to perform quantitative measurements and to try to establish a suitable cut-off point for hemoglobin in urine. The present method is especially suitable for studies of l3TG release in vivo over longer time periods and in settings where blood sampling are impractical, such as ‘Yield” studies. However, it should be noted that plasma measurements of OTG immunoreactivity have draw backs also for short term studies, due to the well-known problems with sampling artifacts. In addition, the long half life of l3TG in plasma (= 100 min; 15) results in a need for long resting periods prior to basal sampling when studying BTG release during physiological interventions in vivo (18). With regard to the problem of how to express data (14), we found similar results whether expressed as concentrations per se or in relation to creatinine excretion (Fig. 1). The latter method, however, seems most attractive to us and eliminates the need for exact estimates of the time during which the urine was produced (expression per unit time) or for quantitative (and often unreliable) 24 h urine collections, which were the methods recommended by Musumeci er al (14). When expressing l3TG excretion per unit cm&nine these problems disappear. In summary, the present assay for selective determinations of HMW OTG in urine which is also based on an improved sensitivity of the radioimmunoassay, provides a much improved
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tool for studies of platelet release reactions in vivo. Our data suggest that physiologically meaningful changes (as exemplified by diurnal variation) may be picked up only when the modified assay is used. The value of selective measurements of HMW BTG immunoreactivity in urine will be determined by further physiological and pathophysiological studies. ACKNOWLEDGEMENTS The study was supported by grants from the Swedish Heart-Lung Foundation and the Swedish Medical Research Council (5930). We are also grateful for receiving partial supplies of materials used to establish sample work-up procedures, i.e. some of the PD-10 columns (Pharmacia, Uppsala, Sweden), some of the Bond-Elut ODS 2Opm columns (Sorbent AB, Vlstra Friilunda, Sweden) and some of the filters used for ultrafiltration from Grace Co. (Mahno, Sweden). G. Oresten of Sorbent AB made helpful suggestions with regard to reverse phase column extractions. The attempts of professor Hans Jomvall, Dept. of Biochemistry, Karolinska Institute, to sequence the BTG-related material in urine are gratefully acknowledged. The expert laboratory assistance of Ms. Caroline Netre is also gratefully acknowledged. REFERENCES 1. LUDLAM, C.A., MOORE, S. BOLTON, A.E., PEPPER, D.S. and- CASH, J.D. The release of a human platelet specific protein measured by a radioimmunoassay. ThrombosisRes. 6: 543-548, 1975. 2. MOORE, S., PEPPER, D.S. and CASH, J.D. The isolation and characterization of a platelet specific O-globulin (&thromboglobulin) and the detection of anti-urokinase and antiplasmin released from thrombin aggregated washed human platelets. Biochim. Biophys. Actu 379: 360-369, 1975. 3. KAPLAN, K.L., NOSSEL, H.L., DRILLINGS, M. and LESZNIK, G. Radioimmunoassay of platelet factor 4 and B-thromboglobulin. Development and application to studies on platelet release in relation to fibrinopeptide A generation. Br. J. Huemufol. 39: 129-146, 1978. 4. KAPLAN, K.L. and OWEN, J. Plasma levels of platelet secretory proteins. CRC Crir. Rev. Oncol. Hematol. 5: 235-255,1986 5. BEGG, G.S., PEPPER, D.S., CHESTERMAN, C.N. and MORGAN, F.J. Complete covalent structure of human l3-thromboglobulin. Biochemistry27: 1739-1744,1978. 6. BURROWS, A.W., CHAVIN, S.I. and HOCKADAY, T.D.R. thromboglobulin concentrations in diabetes mellitus. Luncet i: 235-237,1978.
Plasma
S-
7. VAN OOST, B.A., VELDHUYZEN, B. TIMMERMANS, A.P.M. and SIXMA, J.J. Increased urinary O-thromboglobulin excretion in diabetes assayed with a modified RIA kit technique. Thromb. Haemost. 49: 18-20, 1983. 8. CELLA, G., ZAHAVI, J., DE HASS, H.A. and KAKKAR, V.V. D-Thromboglobulin, platelet production time and platelet function in vascular disease. Br. J. Huemafol. 43: 127-136, 1979. 9. BOLTON, A.E., COOKE, E.D., LEKI-IWANI, C.P. and BOWCOCK, S.A. Urinary l3thromboglobulin as a diagnostic marker for post-operative deep vein thrombosis. Thrombosis Res. 19: 249-255, 1980. 10. DE BOER, A.C., TURPIE, A.G.G., BUTT, R., ZIELINSKY, A. and GENTON, E. Plasma and urine D-thromboglobulin concentration in patients with deep vein thrombosis. Blood 58: 693-698, 1981.
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11. DOYLE, D.J., CHESTERMAN, C.N., CADE, J.F. MeGREADY, J.R., RENNIE, G.C. and MORGAN, F.J. Plasma concentrations of platelet specific proteins correlated with platelet survival. Blood 55: 82-84, 1980. 12. BOLTON, A.E., LUDLAM, C.A., MOORE, S. PEPPER, D.S. and CASH, J.D. Three approaches to the radioimmunoassay of human B-thromboglobulin. Br. J. Haemarol. 33: 233239, 1976. 13. HOPPER, A.H., TINDALL, M. and DAVIES, J.A. Urinary l3-thromboglobulin correlates with impairment of renal function in patients with diabetic nephropathy. Thromb. Haemost. 56: 229-231, 1986.
14. MUSUMECI, V., ROSA, S., CARUSO, A., ZUPPI, C. Urine B-thromboglobulin concentration ratio in single voided urine samples cannot be thromboglobulin excretion. Thromb. Haemost. 55:
ZAPPACOSTA, B., TUTINELLI, F. and or l3-thromboglobulin/creatinine excretion reliably used to estimate quantitative B2-5, 1986.
15. DAWES, J., SMITH, R.C. and PEPPER, D.S. The release, distribution, and clearance of human &thromboglobulin and platelet factor 4. ThrombosisRes. 12: 851-861, 1978. 16. HOLT, J.C., HARRIS, M.E., HOLT, A.M., LANGE, E., HENSCHEN, A. and NIEWAROWSKI, S. Characterization of human platelet basic protein, a precursor form of low-affinity platelet factor 4 and 8-thromboglobulin. Biochemistry25: 1988-1996, 1986. 17. TOFLER, G.H., BREZINSKI, D., SCHAFER, A.I., CZEISLER, C.A., RUTHERFORD, J.D., WILLICH, S.N., GLEASON, R.E., WILLIAMS, G.H. and MULLER, J.E. Concurrent morning increase in platelet aggregability and the risk for myocardial infarction and sudden cardiac death. New Engl. J. Med. 316: 1514-1518,1987. 18. LARSSON, P.T., HJEMDAHL, P., OLSSON, G., EGBERG, N. and HORNSTRA, G. Altered platelet function during mental stress and adrenaline infusions in humans: evidence for an increased aggregability in vivo as measured by filtragometry. Clin. Sci. 76: 369-376,1989.
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