Feb 27, 1984 - 2Department ofMedicine, Harvard Medical School, Boston, MA. 02115. 3Current ... was terminated by extensive dialysis against H20 and lyophilization ..... can readily be learned by a skilled technician and can be rigorously ...
CLIN. CHEM. 30/10, 1692-1696(1984)
ModifiedRadoassay for MeasuringAsialoglycoprotein in Serum Randal Bym,
Peter Thomas,1’2’4 Paul Medrek,’ Zachary Spigelman,’
Asialoglycoproteins are removed from the circulation by the carbohydrate-specific hepatic binding protein in the rat. Asialoglycoprotein in human serum can be detected by an inhibition assay in which the binding of 125l-labeled asialo-a1-acid
glycoprotein (ASAG) to purified hepatocyte membranes is inhibited by known amounts of ASAGor patient’s serum. We were able to reproducibly measure inhibition equivalent to that of 1 to 20 ng of ASAG. The mean concentration of inhibitor in 11 healthy control patients was equivalent to 0.153 (SD 0.051) mg of ASAG per liter, measured in 20 &L of serum. Significant increases of inhibitorwere observed in patients with malignant extrahepatic biliaryobstruction, alcoholic liver disease, viral hepatitis, or liver metastases, but not in those with benign biliaryobstruction. For a set of six control sera the intra- and interassay CVs were 9.1% and 25.2%, respectively (n = 3 each). AddItIonal Keyphrases: liverdisease membraneglycoprofeins hepaticreceptors . proteins cancer referenceinterval The liver plays a central role in the synthesis, clearance, and catabolism of circulating glycoproteins in plasma. The discovery of a hepatic receptor (hepatic binding protein, HBP)5 for desialylated glycoproteins has led to investigation of the carbohydrate-mediated endocytosis system of the liver (1, 2). Sensitive competitive-inhibition assays have been based on the interaction between a test ligand and HBP, involving either intact hepatocyte membranes (3, 4) or a soluble receptor (5). Such tests have been used to detect asialoglycoproteins in serum (6-8) and to evaluate patients with impaired liver function (6-12). Because of our observations of increased concentrations of carcinoembryonic antigen (CEA) in benign liver disease (13, 14) and the demonstration that purified asialo-CEA binds HBP in the rat (15), we wanted to study how factors affecting concentrations of asialoglycoproteins (ASGP) would also affect CEA concentrations. Here we describe a clinically useful assay for HBP-inhibitor (HBP-I) and some early results with it.
Materials and Methods Preparation ofasialo-a1-acidglycoprotein: We purified a1acid glycoprotein by the method of Whitehead and Sammons (16), as modified by Van Lenten and Ashwell (17). To
‘Mallory Gastrointestinal Laboratory, Mallory Institute of Pathology, Boston City Hospital, 784 Massachusetts Ave., Boston, MA 02118. 2Department of Medicine, Harvard Medical School, Boston, MA 02115. 3Current address: Laboratory of Tumor Virus Genetics, DanaFarber Cancer Institute, 44 Binney St., Boston, MA 02115. 4Address correspondenceto this author. Nonstandard abbreviations: ASGP, asialoglycoprotein; ASAG, asialo-a,-acid glycoprotein; HBP, hepatic binding protein; HBP-I, hepatic binding protein inhibitor, BSA, bovine serum albumin; CEA, carcinoembryonic antigen; azpss, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. Received February 27, 1984; acceptedJuly 27, 1984. 1692
CLINICAL CHEMISTRY, Vol. 30, No. 10, 1984
and Norman Zamcheck”2
a pool of outdated plasma (approximately 1 L) we added 2 NIH units of thrombin per milliliter of plasma, to cause it to clot, then performed all subsequent steps as described previously (17). The resulting a1-acid glycoprotein (700 mg) was dialyzed against water and lyophilized. Analysis of 20 pg of the glycoprotein by sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed only a single species after staining with Coomassie Brilliant Blue dye. To prepare asialo-a1-acid glycoprotein (ASAG), we dissolved 20 mg of a1-acid glycoprotein in 2 mL of H20, added 1 U of neuraminidase from Vibrio cholerae (Calbiochem, La Jolla, CA), then incubated for 18 h at 37 #{176}C. The digestion was terminated by extensive dialysis against H20 and lyophilization, after which no neuraminidase activity could be detected. Carbohydrate analysis by gas-liquid chromatography (by Dr. Roger Jeanloz, Massachusetts General Hospital, Boston, MA) revealed no remaining sialic acid residues. We dissolved the lyophilized ASAG in water to prepare a standard solution of ASAG (however, all concentrations reported refer to the dry weight of lyophilized glycoprotein). Aliquots of standard ASAG solution were stored at -70 #{176}C, thawed, used for one week, then discarded. 125I..labeled ASAG was prepared by a modification of the Chloramine-T method of Greenwood et al. (18). The iodinatedglycoprotein was separated from free 125j by gel ifitration on a prepacked Sephadex-G-50 column (Pharmacia, Piscataway, NJ) equilibrated in potassium phosphate (50 mmol/L) containing 1 g of bovine serum albumin (BSA, Cohn Fraction V; Sigma Chemical Co., St. Louis, MO) per liter. More than 95% of the protein in the 4-mL void-volume poo1 was precipitable with trichioroacetic acid; the pools were stored at 4#{176}C until used. The specific activity of the product was 6 to 10 CiJg; we used the label without addition of carrier ASAG except where indicated. Preparation of rat liver membranes: To prepare and fractionate rat liver homogenates, we modified the procedure of Neville (19), doubling all volumes and omitting the final sucrose-gradient ultracentrifligation step. Sucrose concentrations were determined with an Abbe-type refractometer and corrected for temperature effects. The tan-colored membrane floating above the 42.3% sucrose solution was collected with a spatula and washed twice by centrifuging at 100 000 x g for 1 h in 1 mmol/L NaHCO3 solution. Using a Dounce homogenizer, we suspended the final pellet in the wash buffer, then prepared aliquots and stored them at -70 #{176}C. The yield of protein, determined by the procedure of Lowry et al. (20), averaged 1.83 mg/g wet wt. of liver for three separate membrane preparations. The membranes were thawed at 20 #{176}C before use. Membrane binding assay: The assay conditions are slightly modified from the procedure of Van Lenten and Ashwell (4). Each 12 x 75 mm polypropylene tube contains 50 &L of buffer A [per liter, 2 mol of NaC1,100 mmol of CaCl2, 1 g of BSA, and 20 mniol of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.5]; 150 p.L of buffer B [per liter, 20 mmol of HEPES, 1 g of BSA, pH 7.4]; 100 L of membranes diluted in buffer B; 100 L of H2O or standard ASAG solution in H2O; and 200 ng of 1I-labeled ASAG in 100 L of buffer B.
To determine the ASAG-binding capacity of each membrane preparation, prepare a series of tubes containing from 0 to 1 mg of membrane protein, and either H20 or 20 pg of ASAG for the zero inhibitor (B0) or nonspecific binding control tubes, respectively. Incubate for 1 h at 37 #{176}C with vigorous agitation, then stop the reaction by immersing the tubes in an ice bath. Add 2 mL of buffer C (per liter, 0.1 mol of NaC1, 10 mmol of Tris, 0.1 mol of CaCl2, and 1 g of BSA, pH 7.5) at 0#{176}C to each tube and collect the membranes on GF/C ifiters (Whatman Laboratory Products, Clifton, NJ) under reduced pressure. Count the radioactivity on the ifiters (we used an Autogamma 500C from Packard Instruments, Downers Grove, IL), and determine the amount of ‘I-labeled ASAG absorbed to the membrane by subtracting the nonspecific binding counts from those for the B0 sample at each concentration tested. Tests of nonspecific binding of ‘I-labeled ASAG to GF/C ifiters in the absence of membrane indicated that the background binding could be reduced by 80% by preincubating the filters in BSA (50 g/ L of buffer C) for 30 mm before the assay. Membrane-binding inhibition assay: The two-stage inhibition assay is also a modification of the previously described method (4). First, mix 50 L of buffer A, 150 L of buffer B, 100tL of H2Oor standard ASAG inhibitor in H2O, and 100 pg of membrane protein in buffer B, then incubate for 1 h with vigorous agitation. Next, add 100 tL (200 ng) of ‘I-labeled ASAG and continue the agitation for another hour. Terminate the reaction by plunging the samples into an ice bath and determine the membrane-bound radioactivity by ifitering with GF/C filters as described above. Prepare a standard curve by testing 0.02, 0.2, 2.0, 4.0, and 20 ng of ASAG as inhibitors. Include in each assay positive controls containing H20 (B0)and negative controls containing 20 pg of ASAG (for nonspecific binding) as inhibitors. The order of samples being filtered (21) or the duration of the ice-bath incubation (up to 2 h) had no detectable effect on the results. To test for asialoglycoproteins in patients’ or control sera, add 20 pL of serum to a tube containing no other inhibitor and follow all procedures exactly as for the standard curve. Test all samples, serum or standard, in duplicate and use the average bound counts/mm to calculate, by automated linear interpolation, the concentration of the unknown from the standard curve. The calculated result, representing inhibition in 20 iL of serum equivalent to that from known nanogram amounts of ASAG, is then converted to units of ASAG-mg-equivalents per liter of serum. Serum samples: We studied 45 patients treated at Mount Auburn Hospital in Cambridge, MA, and at Dana Farber Cancer Institute, Boston, MA. The 20 women and 25 men ranged in age from 29 to 85 years. Nine patients had malignant extrahepatic biliary obstruction (eight with pancreatic cancer and one with cholangiocarcinoma); five had benign extrahepatic biliary obstruction; seven had viral hepatitis; nine had alcoholic liver disease; and 15 had liver metastases (nine colon, three lung, one cholangiocarcinoma, one bladder, and one unidentified epithelial carcinoma). Informed consent was obtained from all participants. The normal control group consisted of laboratory personnel (three women and eight men, ages 21 to 42 years). Serum samples were collected in Vacutainer Tubes and the serum stored at -20 #{176}C. Liver function tests and measurement of serum bilirubin, alkaline phosphatase, aspartate aminotransferase, and CEA were performed on the same blood samples analyzed for HBP-I.
Results Binding
experiments
we incubated increasing amounts of purified plasma membranes with a constant amount of labeled ASAG. Bound radioactivity varied linearly with the amount of membrane preparation over the range 50 pg to 1 mg of membrane protein, with a slope of 3.3 ng of ASAG bound per 100 pg of membrane protein. Therefore, we used a standard quantity of 100 pg of membrane protein per test. Comparison of inhibition produced by serum inhibitors and purified ASAG: To determine the approximate concentration of HBP-I in serum and to test for nonspecific serum effects on the assay, the inhibition curve produced by serial dilutions of a control subject’s serum was compared with that obtained with purified ASAG (Figure 1). The B0 appeared to be the same and the shapes of the curves were similar, both consistent with the hypothesis that the serum components and the ASAG interact with the same receptor. Experiments in which aliquots of a single serum sample were tested after having been supplemented with known amounts of purified ASAG demonstrated that components in 20 L of serum had no effect on nonspecific binding (results not shown). Standard curve of the assay: Various amounts of ASAG were added to 100 pg of membrane proteins; the standard curve was reproducible over the range 0 to 20000 ng. Figure 2 shows the average of 12 standard curves. TheB0averaged approximately 3% of the total lasl.labeled ASAG added and the radioactivity for the 20000-ng point (nonspecific binding) averaged 10% of the B0 value. Inter- and intra-assay variation of normal controls: A series of six normal control sera, obtained from laboratory personnel with normal liver-function test profiles, were tested on three consecutive days. From the results of duplicate determinations (Table 1) the average intra- and interassay CVs were 9.1% and 25.2%, respectively. Intra -assay variation of patients’ samples: The first results for patients’ samples tested that fell into the HBP-I ranges of 0-0.20,0.20-0.40, 0.40-0.60, and 0.60-0.80 mg/L were compiled until we had 10 samples in each group (only five samples were available in the 0.60-0.80 mg/L group). The #{163} utof nfl
60
fl1
I
80
serum (log sca’e) 1
I
10
100
T
I
-
C
40 (..J #{149}1
20
I 0
‘#{176}
I 0.2
I
I
I
2.0
20
200
2000
ng ASAG(log scale) capacity
of rat liver
membranes:
In a series of
to establish optimum conditions for the assay,
Fig. 1. ASAG binding to liver membranesas inhibitedbypurified ASAG (#{149}) or control serum (A) CLINICAL CHEMISTRY, Vol. 30, No. 10, 1984 1693
for asialoglycoproteins in serum is specific, sensitive, reproducible, and clinically useful. We selected from reportassay
100
-
80
-
60
-
40
-
ed assay methods (3,6, 7) the conditions moat appropriate for our needs. The two-step procedure, in which inhibitor was first incubated with the receptor and later incubated with a near-saturating amount of radiolabeled test ligand, was selected because of its sensitivity. In preliminary experiments we achieved about a 10-fold increase in sensitivity over that of the one-step competitive assay (7). The operational irreversibility of the binding of ASAG to membranes under the conditions of the assay was demonstrated by the stability of the receptor-ligand complexes on ice for upto2h.Theuseofsmallserumsamples(20L)allowedus
to repeatedly test valuable patients’ samples over a period of time. Tests of control samples that were frozen and thawed 20
#{149}
as many as five times showed a maximum variation of 7%. This stability of values obtained from frozen samples increases the assay’s clinical utility.
20,000(NSB)
The variation observed between assays is probably related to the use of liver membrane fragments as the immobilized HBP receptor. Although every effort was made to
-
0.02
0.2
i i
i
2.0 4.0
20
n ASAG Inhibit
r
Fig. 2. Standard curve for HBP-l assay, determined as counts/mm bound in the presence of inhibitor (B) divided by counts/mm bound in no-inhibitor controls (B0) The dashed lines representthe 95% confidenceintervalof the meanfor the 12 setsof duplicatedeterminationsof standards(7). The 20000 ng pointrepresents nonspecific binding _____________________________________
Table 1. HBP-i Concentrations in Six Normal Control Sera HBP I -,
L
Day1 0.188
Day 2
Day3
0.149
0.i
o.i
0.178
0.133
0.159
(0.020)
0.155
0.133
0.280
(009
0.163
0.141 0.131
0.209
0.182
0.216
0.174 (0.035)
0.094 0.097
0.058 0.064
0.115 0.060
0.087 (0.022)
0 174 0:181
0 121 0:140
0 0:177
0 169 (0:035)
Mean (SD)
0.171 0.090 0.168 0.168 0.089 0.216 Duplicatedeterminationson each of three days.
0.151 (0.051)
CV for duplicate determinations within these groups was 4.9%, 4.1%, 6.1%, 10.0%, and 3.5%, respectively. HBP-I concentrations in controls’ and diseased patients’ samples: After grouping patients by diagnosis on the basis of laboratory and clinical information, we tested the first sample available from each patient. The 11 normal controls had a mean HBP-I concentration of 0.153 (SD 0.051) mg/L. The mean SD for the patient groups (mg/L) were: for five benign biliary obstruction patients, 0.173 ± 0.042; nine with malignant biliary obstruction, 0.384 ± 0.259; seven with viral hepatitis, 0.230 ± 0.065; nine with alcoholic liver disease, 0.379 ± 0.148; and 15 with liver metastases, 0.432 ± 0.286. Analyses of serial samples from these patients will bethe subject of a later report.
Discussion This competitive-inhibition 1694
membrane
receptor-binding
CLINICAL CHEMISTRY, Vol. 30, No. 10, 1984
suspend and aliquot the membranes in homogeneous suepensions, it was not possible to eliminate microaggregation. We feel the precision achieved with the assay, when considered in relation to the range of values associated with the clinically important disease groups, is sufficient to provide useful clinical information. HBP is found in substantial quantities in most membrane fractions prepared from rat liver by standard fractionation schemes (23). We quantified membrane HBP activity under conditions in which the percentage of radioactive ASAG bound varied from 0.1 to 5%, and observed an almost linear relationship between the quantity of membranes and the ASAG bound. The activity of membranes we prepared was lower than that reported by Van Lenten and Ashwell (4). We selected a method of membrane purification that, though it requires an ultracentrifuge and a refractometer, can readily be learned by a skilled technician and can be rigorously controlled. We have investigated more simple methods of membrane fractionation (24) but were unable to overcome problems of high nonspecific binding. We observed no loss of binding activity of membranes stored at -70 #{176}C for as long as three months. We selected ASAG as the test ligand because of its high binding affinity for HBP and because we specifically wanted to search for inhibitors in serum capable of blocking another high-affinity binder, asialo-CEA. This test ligand also has the advantage that it can readily be prepared from pooled human serum but is also available from several commercial sources. The demonstration of high-affinity inhibitors in serum is a starting point for further study. The results for control subjects reported here, 0.153 ± 0.05 1 mg ASAG equivalents per liter of serum, are lower than the values of 0.758 ± 0.350 mg desialylated thyroxinbinding globulin equivalents per liter reported by Marshall et al. (11) or of 0.324 ± 0.091 mg ASAG equivalents per liter reported by Arima (10). Marshall et al. showed that desialylated thyroxin-binding globulin binds to hepatocyte membranes with about 10-fold less affinity than does ASAG. Their use of a lower-affinity test ligand would be expected to generate higher serum inhibitor values if the inhibitory activity in serum has an affinity greater than or equal to that of the test ligand. The reason for the difference between the control values of the Arima group and ours probably is related to the single-step, nonsaturated assay used by Arimaetal.(7). The mechanism of production of asialoglycoproteins in humans is unknown. The theory that ASGPs are generated
from normal glycoproteins (1,2,25) has been challenged by a comparison of clearance rates, in vivo, for fetuin in the presence and absence of saturating amounts of ASGP (26). The report that polymeric IgA1, which normally expresses terminal galactose residues, binds to HBP indicates another possible source of ASGP (27). Further qualitative and quantitative analysis of ASGPs is needed to explain their presence in the circulation. The ASGP receptor, estimated at 500 000 copies per rat hepatocyte and capable of ligand internalization at a rate of 0.1 pmollmin per 106 cells, has been extensively studied in vitro (28,29). These studies were extended by Pardridge et al. (30), who reported an in vivo Km of 260 mgfL for ASAG in
rats. Studies in rats have also shown that HBP concentration and HBP-I activity can be modified under various pathological conditions (31-35). The normal concentrations of ASGP observed in the present study, about 0.150 mg/L in human serum, are more than 1000-fold lower than the half-saturating concentration observed in rats. On the basis of this comparison, we conclude that HBP-I content would have to be greatly increased to inhibit significantly the asialoglycoprotein binding activity of the normal liver. Measurements of HBPI in human liver tissue (36, 37) and measurements of the total ASGP binding capacity of the liver, under normal and pathological conditions, are needed for evaluating the contribution of tissue variation in concentrations of receptor to the concentrations of HBP-I in patients’ livers. If decreased receptor activity is a major cause of increased serum concentrations of HBP-I, then this decrease must be severe indeed to account for the failure to clear such small amounts of HBP-I
from the circulation.
The availability of a sensitive assay for ASGPs will allow the further study of ASGP concentrations in the sera of patients with benign and malignant diseases. The very low concentrations of ASGP detected in control patients and the moderately increased concentrations in patients with liver disease suggest that increases in serum ASGP may indicate hepatic injury or increased production of ASGP. The clinical significance of HBP-I concentrations will rely on further study of the relationship between circulating glycoproteins and the liver. Supported by grant no. CA-04486 Institute.
from
the National
Cancer
References 1. Ashwell G, Morel! AG. The role of surface carbohydrates in the hepatic recognition and transport of circulating glycoproteins. Ado Enzymol 41, 99-128 (1974).
2. Morell AG, Gregoriadis G, Scheinberg ff1, eta!. The role ofsialic acid in determining the survival of glycoproteins in the circulation. JBiol Chem 246, 1461-1467 (1971). 3. Pricer WE Jr, Ashwell G. The binding of desialylated glycoprothins by plasma membranes of rat liver. J Biol C/rem 246, 48254833 (1974). 4. Van Lenten L, Ashwell G. The binding of desialylated glycoproteins by plasma membrane
of rat liver. Development of a quantita-
tive inhibition assay. J Biol C/rem 247, 4633-4640 (1972). 5. Hudgin RL, Pricer WE Jr, Ashwell G, et al. The isolation and properties of a rabbit liver binding protein specific for asialoglycoproteins. J Biol C/rem 249, 5536-5546 (1974). 6. Marshall JS, Green AM, Pensky J, et a!. Measurement of circulating desialylated glycoproteins and correlation with hepatocellular damage. J Clin Invest 54, 555-562 (1974). 7. Arima T, Motoyama Y, Nagata K, Kondo T. Serum glycoprotein in liver diseases I. Studies on sialic acid-free glycoproteins. Improved competitive binding assay of desialylated glycoproteins by rat liver plasma membrane. GastroenterolJpn 11, 300-306(1976). 8. Lunney JK. Studies on the regulation
of serum
glycoproteins
homeostasis. Ph.D. dissertation, The Johns Hopkins University,
Baltimore, MD, 1976. 9. Arima T, Motoyama Y, Yamamoto T, et al. Serum dycoproteins in liver diseases ifi. Desialylated glycoproteins in acute hepatitis. Gastroenterol Jpn 12, 39-42 (1977). 10. Arima T. Serum glycoprotein in the liver diseases Vffl. Desialylated glycoprotein in the liver cirrhosis. (JastroenterolJpn 14,349-
352 (1979). 11. Marshall JS, Williams S, Jones P, Hepner GW. Serum desialylated glycoproteinsin patientswith hepatobiliary dysfunction. JLab Clin Med 92, 30-37 (1978). 12. Arima T, Motoyama Y, Yamamoto T, et al. Serum glycoprothins in the liver diseases V. Desialylated glycoproteins in chronic hepatitis. Gastroenterol Jpn 13, 503-506 (1978). 13. Loewenstein
MA, Zamcheck
N. Carcinoembryonic
antigen
(CEA) levels in benign gastrointestinal disease states. Cancer 42, 1412-1418 (1978). 14. Thomas P, Zamcheck N. Role of the liver in clearance and excretion of circulating carcinoembryonic antigen (CEA). Dig Di.s Sci 28, 216-224 (1983). 15. Thomas P, Hems DA. Hepatic clearance of circulating native and asialocarcinoembryonic antigen by the rat. Biochem Biophys Res Commun 67, 1205-1209 (1975). 16. Whitehead PH, Sainmons HG. A simple technique for the isolation of orosomucoid from normal and pathological sera. Biochim Biophys Acta 124, 209-211 (1966).
17. Van Lenten L, Ashwell G. Studies on the chemical and enzymatic modification of glycoproteins.A general method for the tritiation of sialic acid containing glycoproteins. J Biol C/rem 246, 1889-1894 (1971). 18. GreenwoodFC, Hunter WM, GloverJS. The preparationof‘3’Ilabeled human growth hormone of high specific radioactivity. Biochem J 89, 114-123 (1963). 19. Neville DM. Isolation of an organ specific antigen from cell surface membrane of liver. Biochim Biophys Ada 154, 540-552 (1968).
20. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenolreagent.JBiol C/rem 193,265275 (1951). 21. Hung CR, Hong JS, Bondy SC. The preventionof an artifact in receptor binding assay by an improved technique. Life Sci 30, 1713-
1720 (1982). 22. Rogers CRP. Quality Control and Data Analysis in BinderLigand Assay, Scientific Newsletters, Inc., Anaheim, CA, 1981, pp 134-147. 23. Pricer WE, Ashwell G. Subcellular distribution of a mammalian hepatic binding protein specific for asialoglycoproteins. J Biol Chem 251, 7539-7544 (1976). 24. Kamath SA, Narayan KA. Interaction of Ca2 with endoplasmic reticulum of rat liver: A standardized procedure for the isolation of rat liver microsomes. Anal Biochem 48, 53-61 (1972). 25. Bocci V. The role of sialic acid in determining the lifespan of circulating cells and glycoproteins. Experientia 32, 135-140 (1976). 26. Clarenburg R. Asialoglycoprotein receptor is uninvolved in clearing intact glycoproteins from rat blood. Am J Physiol 244, G247-G253 (1983). 27. Stockert RJ, Kressner MS, Collins JC, et al. IgA interaction with the asialoglycoprotein receptor. Proc Nail Acad Sci USA 79,
(1982). 28. Schwartz AL, Rup D, LodishHF. Difficulties in the quantitation of asialoglycoprotein receptors on the rat hepatocyte. J Biol C/rem 255, 9033-9036 (1980). 29. Regoeczi E, Debanne MT, Hatton MWC, Hoj A. Elimination of asialofetuinand asialoorosomucoid by the intact rat. Quantitative aspectsof the hepatic clearancemechanism.Biochim Biophys Acta 6229-6231
541, 372-384 (1978). 30. Pardridge WM, Van Herle AJ, Naruse RT, et a!. In vivo quantitation of receptor mediated uptake of asialoglycoproteins by rat liver. J Biol C/rem 258, 990-994 (1983).
31. Hickman
J, Ashwell G. Studies on the hepatic binding of
asialoglycoproteins by hepatomatissueand by isolated hepatocytes. Enzyme Therapy of Lysosomal Storage Diseases, JM Tager, GJM Hooghwinkel, WTH Daems, Eds., North-Holland Pub!. Co., New
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32. Stockert RJ, Becker FF. Diminished hepatic binding protein for desialylated glycoproteins during chemical hepatocarcinogenesis. Cancer Res 40, 3632-3634 (1980). 33. Osada J, Zawirska B, Chorazyczewski J, Dobryszycka W. Catabolism ofdesialylated glycoproteins during carcinogenesis and inflammation in rats. Acta Biochim Pol 28, 147-156 (1981). 3& Wong MW, Jamieson JC. Evidence for reduced uptake of asialo a1-acidglycoprotein during the acute phase responseto inflammation. Life Sci 25, 827-834 (1979). 35. Sawamura T, Kawasato S, Shiozaki Y, et al. Decreaseof a
hepatic binding protein specific for asialoglycoproteins with accumulation of serum asialoglycoproteins in galactosamine-treated
rats. Gastroenterology81, 527-533 (1981). 36. Arima T, KondoT, Nagashima H. Serum glycoproteins in the liver diseasesVI. The presenceofdesialylated glycoprotein binding activity in particulate fraction of human liver homogenate.Gastroenterol Jpn 13, 503-511 (1978). 37. Baenzinger JU, Maynard Y. Human hepatic lectin. Physiochemical properties and specificity. J Biol C/rem 255, 4607-4613 (1980).
CLIN. CHEM. 30/10, 1696-1700 (1984)
Analysesfor Progesteronein Serum by Gas Chromatography/Mass Spectrometry:Target Data for ExternalQualityAssessmentof Routine Assays Simon J. Gaskell,1 Brian G. Brownsey, and Graham V. Groom We describe a procedure for measuring progesterone in plasma and serum by isotope dilution and mass spectrometry. Extracition with use of a microcellulose-coupled antiserum is followed by conversion to the 3-enol heptafluorobutyrate and analysis by gas chromatography/mass spectrometry (GCIMS) with selected ion monitoring, at a resolution of 5000. Interassay CVs were 1.5 to 5.4% for the concentration range 13 to 43 nmol/L. Analyses of various serum volumes showed excellent linearity. Accurate determination of progesterone added to serum was demonstrated. Plasma and serum pools were analyzed to provide target data for use in the U.K. national external quality-assessment scheme for progesterone assays. Direct, non-extraction radioimmunoassays and those incorporating solvent extraction both showed a positive bias with respect to data obtained by the present procedure, but the bias was more marked with the direct assays. AddItional Keyphrasee: steroids ity studies
ovulationdetection
fertil-
Assays for progesterone in blood plasma or serum are widely used for the detection of ovulation and for investigation of control of the ovarian cycle. Concern over the performance of routine progesterone assays (usually radioimmunoassays) has prompted the establishment of external quality-assessment schemes, in parallel with those operating for other steroids. The principal objective of such schemes is the assessment of relative bias, for which the availability of stable, independent target data is clearly beneficial. Isotope dilution with gas chromatography/mass spectrometry (GC/MS)2 detection is increasingly accepted as a suitable and highly specific reference technique. Previous Tenovus Institute for Cancer Research, Welsh National Schoolof Medicine, Heath, Cardiff CF4 4XX, Wales, U.K. ‘Current address: Laboratory of Molecular Biophysics, NIEHS, P.O. Box 12233, Research Thangle Park, NC 27709. 2Nonsthrd abbreviations: GC, gas chromatography; MS. mass spectrometry; GLTM, group-laboratory trimmed mean; A1Thm, all-laboratory trimmed mean; NEQAS, National External Quality
AssessmentScheme. Received July 17, 1984; accepted July 27, 1984. 1696
CLINICALCHEMISTRY, Vol. 30, No. 10, 1984
work from this laboratory (1,2) and others (3,4) has clearly established an important role for GC/MS target data in external quality-assessment schemes. Accordingly, we have extended our previous work on other steroid hormones (1,2) to the analysis of progesterone. We report here the GC/MS analysis for progesterone in plasma and serum pools that is used in the United Kingdom National External Quality Assessment Scheme (NzqAs).
Materials and Methods Samples Over the period of the study, 22 sample pools were to participants in the scheme. Seventeen pools, covering the range 4-65 nmol/L (as subsequently determined by GC/MS), consisted of sera from male or female donors, without added progesterone. Most of the pools included sera from more than one donor; however, samples from males and females were not mixed. Two further serum pools included a supplement of progesterone (Sigma Chemical Co., Poole, U.K.), giving total concentrations of
