ANALYTICAL CHEMISTRY, VOL. 50, NO. 3, MARCH 1978
449
Radioimmunoassay of Calcitonin in Normal Human Urine Richard H. Snider,' Charles F. Moore, Omega L. Silva, and Kenneth L. Becker Metabolic Research Laboratory (688115 1J). Veterans Administration Hospital, 50 Irving Street, NW, Washington, D.C. 20422
Direct radioimmunoassay of calcitonin in human urines containing 1 3 ng/mL is complicated by interference from substances most probably structurally similar to the methylated xanthines. A simple, reproducible procedure is described for the removal of the interfering substances prior to radioimmunoassay. Utilizing this procedure and a sensitive antiserum specific for the carboxyl terminal region of calcitonin, it has been possible to obtain precise estimates of calcitonin concentrations in the urine over the range, 0.02-3.0 ng/mL within which calcitonin is found in normal urine. The intra- and interassay s/X were 5 and 1 5 % , respectively. The urine calcitonin values apparently reflect serum calcitonin concentrations (e.g., urine/serum r = 0.9873 for 40 hypercalcitonemic patients); but urine calcitonin determination has two important advantages: greater reproduciblllty because of decreased heterogeneity and greater differentiationof patient populations. I n view of these results, the assay of urine calcitonin may prove to be a very useful clinical tool.
Since the discovery of the hypocalcemic, hypophosphatemic polypeptide hormone calcitonin (CT),by Copp ( I ) in 1961, much effort has been directed toward the development of techniques for its measurement in biologic fluids. Much of t h e uncertainty over serum concentrations in normals as determined by radioimmunoassay (RIA) appears to emanate from size- and immuno-heterogeneity of the hormone as well a s the presence of interfering substances (2). In 1971 Voelkel and Tashjian (3) reported finding hypocalcemic activity in the urine of patients with medullary thyroid cancer (MTC). Later Melvin and co-workers (4) found t h a t the hypocalcemic activity in the urine was immunochemically similar to human CT and co-eluted with the synthetic hormone on short G-75 Sephadex columns. W e have developed a simple, reproducible technique for the assay of CT in human urine which should yield consist,ent interlaboratory results provided that the same standards and assay conditions are utilized. Urinary C T concentrations appear to reflect CT concentrations in the serum (particular) for patients with hypercalcitonemia) and they afford an earlier indication of a n increased production rate or metabolic clearance of the hormone, and permit the study of physiologic changes which may not be detected in the serum.
EXPERIMENTAL Collection a n d Storage of Samples. Serum from fasting patients was collected in 13 X 100 mm non-siliconized glass tubes (Venoject; Kimble-Terumo, Elkt>on,Md. 21921) and stored at -20 O C until assayed. Urine from fasting patients was collected in polyethylene or flint glass receptacles containing sufficient NH4HCOrIto maintain a pH 2 7.5. Usually the first morning urine was discarded, and the urine for assay was collected over a fixed period of time (1-2 h). One-mL aliquots of the urine are stored at -20 "C until assayed. Radioimmunoassay of Serum CT. Synthetic human CT (see Acknowledgment) was labeled to a specific activity of 150-250 pCi/pg utilizing a modification of the Hunter- Greenwood chloramine-T method ( 5 ) . Non-equilibrium and equilibrium This paper not subject to U.S. Copyright.
assays were performed with carboxyl terminal antiserum Ab-IV and midportion antisera Ab-I1 and Ab-111 as previously described ( 2 ) . A new midportion antiserum A4h-IIIbvery similar to Ab-111, but 2-3 times more sensitive (based on 50% [B,/B,I in a logit-log plot ot'the standard curve) than Ab-IV was also )utilized. Calculated from Scatchard plots (61,the dissociation constants (KDb) were -3.2 X lo-" M for Ab-I\' and -1.0 X 10." for Ab-IIIb, and the binding site concentrations were -2.3 X lo-" M for Ab-IV and -7.6 X 10 l 2 M for Ab-IIIb. Serum aliquots of' 550 pL were used for most assays. Beckman CT (according to Beckman, their 0.5-mg vial (lot =B0903) contains 0.58 mg human CT) was utilized to generate the data in this paper. This standard yields slightly lower concentrations ( - 2 0 % ) than we have reported in earlier papers using Organon CT standards. Purification of Urine Samples. The 1-mL aliquot for RIA was boiled for 3 min and dextran blue (R-2000,2 000 000 daltons; Sigma Chemical Co., 3500 DeKalb Street, St. Louis, Mo. 63118) added. The boiled urine was passed through a 6 X 220 mm glass column containing 5 mL polyacrylamide gel (Bio-Gel P-2; lO(t200 mesh; Bio-Rad Laboratories, 32nd and Griffin, Richmond, Calif. 94804) suspended in 0.1 h.I KH4HC03at pH 7.S. The blue void volume was collected in a 7-mL non-siliconized glass tube, lyophilized. and reconstituted in 2 mI, of i% (w/v) human serum albumin (Cutter Labs, Inc. Berkeley, Calif. 94710) in buffer containing 0.15M NaCl and 0.13 M H3B03at pH '7.5. Columns were rinsed with 1 M HC1 and repacked before use. Urines containing 210 ng/mL were not purified. Radioimmunoassay of Urine CT. The assay was carried out as described for serum except for the addition of 10% guinea pig or hypocalcitonin human serum. The addition of guinea pig serum accomplished two functions: assay sensitivity was increased (2) and the occasional non-parallelism of dilution curves for urine CT component U-2 as compared to the standard curve was eliminated. The boiled and gel filtered urine which was reconstituted to 2 mL is assayed in aliquots of 10-200 pL. Urine column fractions were assayed as described for serum fractions. Permeation Gel Chromatography. Urine samples were chromatographed on 3 X 110 cm glass columns containing 800 mL G-75 superfine Sephadex suspended in 0.1 M NHIHCOBat pH 7 . 5 or 0.1 hl SH4O2CHat pH 4.7. The sample was eluted in 6.4-mL aliquots (7-8 aliquots/O.l Kd) which were assayed and the concentrations were plotted graphically as described previously for serum ( 2 ) . Concentration of Urine CT for Permeation Gel Chromatography on G-7.5 Sephadex Columns. Concentration of the CT in urine samples containing more than 200 pg/mL was unnecessary; however, the following procedures were utilized on some high CT as well as low CT samples to determine recoveries. Trichloroacetic A c i d Precipitation of Protein. [Trine CT was co-precipitated with urine proteins by the addition of 10% trichloroacetic acid as described by L. Constan and co-workers for proinsulin and insulin (71. Petroieunz Ether Emulsion. Alkaline urine was added to 300 mI, petroleum ether (ACS grade), (bp 35-60 "C, Fisher Scientific CO.. Silver Spring. hld. 20910) in a 2-1, separatory funnel. The funnel was shaken vigorously for 2 min and the two phases were allowed to separate. An emulsion formed at the interface. The ether and aqueous phases were removed and the emulsion was dried by flash evaporation. The dried emulsion, which contained 20-307~ of the total CT, was dissolved in 10 mL 0.1 M NH,HC03 buffer. pH 7 . 5 . and freeze dried. A modification o f the above procedure utilized for urine from some MTC patients was to add 1 mL normal human serum and to reextract following emulsion formation with three more 300-mL portions of petroleum ether. Published 1978 by the American Chemical Society
450
ANALYTICAL CHEMISTRY, VOL. 50, NO. 3, MARCH 1978
Table I. Serum iCT Compared to Urine iCT for MTC Patients Urine iCT
Sample 1
2 3
4 5 6 7
Serum iCT, ndmL 0.19 0.62 0.62 1.2 1.3 1.4
9.0
8
14
9
19 50 50 150
10 11 12
nglmL (A)
3.5 36 2.2 78 46
35 250 210 380 1230 1940 3650
nglplg Creatinine (B) 2.3 15 14
R E S U L T S AND D I S C U S S I O N Radioimmunoassay of CT in Urine. Initially, Melvin and co-workers ( 4 ) indicated that 1 2 5 pL of urine could be radioimmunoassayed directly. In our laboratory, urine samples from MTC and other hypercalcitonemic patients containing 1 3 ng/mL CT did yield results by direct RIA (in aliquots 1 5 pL) which were consistent with results obtained following gel filtration of the urine on our long G-75 Sephadex columns (Figure 1,a and b). The principal fraction of immunoreactive calcitonin (iCT) in most MTC urines, U-2, had an apparent molecular size of 5400 daltons on p H 7.5 columns and 4500 daltons on p H 4.7 columns and was best recognized by carboxyl terminal antisera (3-5 times better than midportion recognizing antisera). The apparent molecular size of U-2 on the p H 7.5 columns is altered following pretreatment with acid or base. Pretreatment of the 5400 dalton fraction with acid yielded the 4500 dalton fraction. Pretreatment of the 4500 dalton fraction with 1 N NHIOH at 55 "C for 1 h yielded a 5400 dalton fraction. Guanidine hydrochloride, 5 M, a t p H 4.7, had no effect on the apparent molecular size of U-2 except the effect described above for low pH. It should be noted that synthetic "'1-HCT added to urine eluted with apparent molecular size of 3500 daltons and its Kd was unaffected by the p H of the columns or pretreatment with acid or base. Sa!mon CT (excreted by patients receiving salmon CT therapy for hypercalcemia) eluted with a Kd much closer to that of synthetic salmon CT monomer. Fraction U-1 (-8000 daltons) was recognized as well or better by midportion antisera as by carboxyl terminal antisera. Because of the greater recognition of the principal MTC urine fraction, U-2, by carboxyl terminal antisera, Ab-IV, a carboxyl terminal antiserum was chosen for most of our urine studies. Although most normal urines tested in our laboratory produced only 5 1 0 % damage of '251-HCT in 24 h (as determined by binding to excess antibody), NH4HC03was added to all urines in order to prevent the degradation of urine CT which occurs a t low pH (3). Basal and post stimulation concentrations of urine CT were correlated better with serum CT concentrations when the urine C T was expressed as ng CT/mg creatinine (Table I). The correlation, r , for 40 basal urine/serum concentrations for MTC patients was 0.9873.
15 24 23 32 20
18
58 3.5 65 35 25 28 15 20 25 39 24
38
26 26 95 480 360 8 30 1020 3950
Mean (t S.D.) Correlation ( r ) This procedure recovered 5570 of the urine CT. Creatinine Determinations. Creatinine was assayed in aliquots of urine as described by Henry (8). Isoelectric Focusing. Sucrose density gradient isoelectric focusing was carried out in various pH ranges at -20 "C on a 110-mL LKB 8101 column (LKB-Produkter AB, Bromma 1, Sweden) essentially as described by Haglund (9).
Ratio (B)/serum
Ratio ( A)/serum
19 11
34 19 17
20 26 22 +7 0.9897
28 i 18
0.9815 860
720
-
- Ab - I1 _ _ _ Ab - IV
600
.-D
E
5c
480
360
u X
240
120
ie
I 0
0.0 0.1 0.2
0.3 0.4 0.5 0.6 0 7 0.8 Urinary HCT for MTC Patient - I P F
0.9 1.0 Kd
1
:I 50
U
lo-
g
r
1 0
00
>
Y I
01
02
I
5
'
I
I
0 4 0 5 0 6 07 0 8 Urinary HCT for MTC Patient E G
03
I
09
10 Kd
Figure 1. (a) Urine from MTC patient having mostly U-2 fraction of calcitonin. (b) Urine from MTC patient having mostly U-1 fraction of
calcitonin Despite the good results obtained for direct RIA of CT in the urine of patients with hypercalcitonemia, difficulties were encountered in trying to determine the CT concentrations in the urine of normals and patients who had had thyroidectomy (Thx). Direct RIA of urines from these patients in aliquots
ANALYTICAL CHEMISTRY, VOL. 50, NO. 3, M A R C H 1978
o'601
"[ 98
a
451
AbIV
- Ab - II
0.50
_ - - Ab - IV
-
cm
c
0
Q
It
0.10
e-.-e 0-0
0.1
0.:2 0 3 0 4 0 5 06 0 7 08 Urinary HCT for Normals Unconcentrated
09
e-*
A -
ll
2
10
20
100 200
50
1
-
-
500 1000 2000
I
000020000500010002 0 0 0 5 0 0 1 002
005
01
02
Aliquot mll
"[ 98
-
b
-
4.8
-Ab - Ill
I
?
M
5
HCT (mi
-
.-
Paraxanthine 1 mg ml Caffeine 1 mg ml
-HCT
-0--@
1.0 Kd
6.0 -
-C
\\
Creal nine 1 mg ml Normal Urine
e--- e Theophylline 1 mg ml
- b
7.2
e
20 -
~~
0.0
-
8.4
70 605040 30 -
- _ _ - Ab - IV
-
-
3.6 I V
,
2.4
-
1.2
-g -1 4-h
I
1;
Urinary Calcitonin in Normal Man (After Concentration1
Figure 2. (a) Unconcentrated urine of a normal person. (b) Concentrated
urine calcitonin (trichloroacetic acid) of a normal person from urine utilized for 2a Table 11. Compounds Interfering with RIA of iCT with Carboxyl Terminal Antisera
Interfering compound Caffeine (1,3,7-trimethylxanthine) Paraxan thine (1,7-dimethylxanthine) Theophylline (1,3-dimethylxanthine) Theobromine (3,7-dimethylxanthine) 7-Methylxanthosine Guanosine Creatinine DNA Deoxyadenosine Thymidine Urea Xanthine Uric acid Bilirubin Proline Histidine Creatine Hemocyanin (Keyhole Limpet)
Apparent iCT in ng iCT/mg interfering compound 2.9 2.1 1.5
0.9 0.9 0.3 0.1
0.1 0.06 0.05 Does not interfere at < 5 0 p L urine Mostly insoluble; interferes slightly Mostly insoluble; interferes slightly Mostly insoluble; binds label 0 0 0 0
2-
u
Figure 3. (a) Dilution curves for interferingsubstances compared to the standard curve in the radioimmunoassay. (b) Dilution curves for U-I, U-2 (without hypocalcitonin serum), and a diluted urine (PF) which had high concentration of iCT compared to the standard curve
of lG20 pL did not correlate well with results for gel filtration of the unconcentrated urine on our long G-'75 Sephadex columns. As shown in Figure 2, a and b, two fractions of C T were found in the urine of normals which corresponded both in apparent molecular size and antibody recognition to those found in MTC patients; however, the total amount obtained for the gel filtration fractions from normals did not correspond to the amount found by direct RIA. The average displacement of IZ5I-HCTfrom antibody for 20-wL aliquots of urine from 23 normals was 25.2% (7' B/B, = 74.8 f 8.4), which would correspond t o a mean C T of 0.44 ng/mL. T h e average displacement of "'I-HCT from excess antibody added a t the end of the routine RIA incubation period was 2.9?& ( % B/Bo = 97.1 h 2.2) indicating t h a t damage to label did not account for much of the observed displacement. Dextran-coated charcoal (10) extracted urine, in 20-pL aliquots, displaced only 8.6% ( % B/Bo = 91.4 & 7.2) IZ5I-HCTfrom antibody in routine RIA; therefore, most of whatever interfered in the RIA as well as the C T was extracted. Chromatography on the long G-75 Sephadex columns revealed t h a t the source of interference was of small molecular size (eluting in t h e salt fraction). A series of known constituents of the urine as well as analogues were tested in the RIA system with the results shown in Figure 3a and Table 11. I t should be noted that the
452
ANALYTICAL CHEMISTRY, VOL. 50, NO. 3, MARCH 1978
urine in Figure 3a was found to contain only 0.035 ng/mL of actual CT. Interestingly, the methylated xanthine, caffeine, was t h e most potent interfering substance producing significant displacement of lZ5I-HCTfrom antibody even at the 500-ng level. Since caffeine and other methylated xanthines are contained in many food and beverage products and are excreted with their metabolites in the urine ( I I ) , these compounds may account for much of the interference. Chloroform removed some, but not all, of the interfering substances from the urine. The interference produced by the methylated xanthines is region specific, affecting carboxyl terminal b u t not midportion recognizing antisera. T h e methylated xanthines and creatinine interfered in equilibrium as well as non-equilibrium assays. Figure 3b gives dilution curves for U-1, U-2 (without hypocalcitonin serum), and MTC urine as compared to the standard curve. R a d i o i m m u n o a s s a y of CT in Gel F i l t e r e d Urine. In order to assay small amounts ( 5 3 ng/mL) of C T in the urine by RIA, a method for separating C T from the interfering substances was developed which satisfied 4 objectives: (1)the method was rapid and simple; (2) the recovery was 290%; (3) replicate samples and identical antisera yielded reproducible results; (4) and the levels determined on purified samples corresponded to the amounts found in C T fractions for the same urine samples on long Sephadex columns. Simkin (12) had previously reported using the affinity of purines for polyacrylamide gel to separate serum uric acid on Bio-Gel P-2 columns. Because the interfering substances were of small molecular size and were likely to be structurally similar to t h e purines, the gel filtration procedure described in t h e Experimental section was devised. When “C-labeled caffeine was added to urine
