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Automated Method for Determining Serum Thiocyanate_ to Distinguish

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Automated Method for Determining Serum Thiocyanate_ to Distinguish Powered By Docstoc
					CLIN. CHEM. 20/10,            1344-1348      (1974)




Automated Method for Determining Serum Thiocyanate,

to Distinguish Smokers from Nonsmokers

William C. Butts, Martha Kuehneman, and Graham M. Widdowson


A chemical        test to distinguish smokers and nonsmokers                                  after cessation of smoking. In contrast, serum or plas-
is important       in many epidemiologic   studies. We have de-                               ma thiocyanate     concentrations   have a half-life of
veloped        an automated method for determining serum                                      about 14 days (6) and thus give a more practical mdi-
thiocyanate by use of the reaction between thiocyanate                                        cation of a person’s smoking habits from an epide-
and ferric ions to form a colored complex. Our data                                           miologic     point      of view.   For    application     to the   large
show that this method distinguishes cigarette smokers
                                                                                              number of specimens from an epidemiologic           study,
and nonsmokers: of 197 healthy individuals studied,
                                                                                              an analytical method should be easily automated        and
1.8%         false-positive        and 6.7 % false-negative                        results
were obtained when 85 smoI of SCN                                per liter was the            relatively   inexpensive   to perform; in addition,     the
critical value used to distinguish the two populations.                                       sample required should be easy to collect, store, and
                                                                                              transport    before analysis. The preceding     consider-
AdditIonal        Keyphrases:         calorimetry                S
                                                            #{149} Ct’t     -Fe3      reac-   ations led us to conclude that serum or plasma thio-
tion       #{149}AutoArzalyzer                                                                cyanate     is the       determination        of choice      in such       a
                                                                                              study.
     With      the implication            of cigarette         smoking          as a risk        Colorimetric   methods based on two different prin-
factor in lung cancer, emphysema,      and coronary                                           ciples have been used for the manual determination
heart disease, many epidemiologic    studies include                                          of thiocyanate    in biological fluids. In the method of
programs designed to encourage participants  to stop                                          Aldridge (10) cyanide and thiocyanate        are converted
smoking.         To accurately    evaluate    the data from these                             to cyanogen bromide by the addition             of bromine
studies,       some corroboration       is needed of the partici-                             water, and a colored complex is formed between the
pants’       verbal    assurance           that they have decreased or                        cyanogen bromide and a pyridine-benzidine-hydro-
ceased their smoking.                     Eight of 107 persons in one                         chloride mixture. In the method described by Bowler
smoking           withdrawal         program          who      claimed          to have       (11), thiocyanate     in protein-free   serum filtrates is
stopped  smoking had not actually done so (1).                                                reacted with ferric nitrate to produce ferric thiocy-
   Many workers have shown that both serum and                                                ante, which is highly colored. Van Peborgh (12) and
plasma thiocyanate    (2-6) and blood carboxyhemo-                                            Pettigrew and Fell (6) have recently used modifica-
globin (1, 6-8) concentrations  are higher in smokers                                         tions   of Aldridge’s      method;       the former     is an automat-
than in nonsmokers     and that either determination                                          ed method in which 3-methyl- 1-phenyl-5-pyrazoline
can be used to detect whether or not a person is                                              is used as the color reagent instead of the carcinogen-
smoking.          Carboxyhemoglobin                   has     a half-life          of ap-     ic benzidine; the latter is a manual method in which
proximately  4 h (9), so that its concentrations     in                                       the benzidine is replaced by p- phenylenediamine.     All
blood remain supranormal   for a relatively short time                                        the procedures    based on Aldridge’s method involve
                                                                                              several different reagents and are more expensive to
                                                                                              automate than is Bowler’s method. We therefore de-
   Institute      of Health      Research,     P.O. Box 7999,             San   Francisco,
Calif.   94120.                                                                               cided to adapt and optimize the reaction of ferric ni-
   Received June 24,          1974; accepted    Aug. 6, 1974.                                 trate with thiocyanate   on the AutoAnalyzer.

1344       CLINICALCHEMISTRY,                Vol. 20, No. 10, 1974
Materials and Methods
                                                                                                  Table 1. Influence of Diluent Composition on
Reagents                                                                                                 Recovery of SCN from Serum
   Stock            thiocyanate                   imol of SCN
                                              standard             (1000     Diluent
                                                                               salt
per liter): Dissolve 97.2 mg of potassium thiocyanate                        concn,              Dlluent             Standard        Recovery,
                                                                            mol/liter               salt               matrix            %
in de-ionized water and dilute the solution to 1 liter
                                                                               0.15              NaCI                  H20                87
with water. It is stable at room temperature                for three
                                                                               0.1               NaCI                  H20                86
months.
    Working    thiocyanate       standards     containing 20, 50,              0.15              NaCl                  BSA5               95
                                                                               0.1               NaCIO4                H20               101
100, and 250 imol per liter, respectively,            are made by
                                                                               0.2               NaClO4                H20               100
appropriately      diluting the stock thiocyanate            solution
                                                                               0.4               NaCIO4                H20               100
with water. Prepare every two weeks; store at room
temperature.                                                                 Recovery     based      on 150 zmol of SCN        per liter added to
   Ferric nitrate      reagent:     Dissolve 10.0 g of ferric ni-        pooled serum containing 60 imol of SCN per liter.
                                                                           S Bovine   serum     albumin,   30 g/liter.
trate[Fe(NO3)3       - 9H2O] in nitric acid (1.5 mol/liter)
and dilute to 1 liter with 1.5 mol/liter nitric acid. Fil-
ter and store in a dark bottle at room temperature;
prepare every two weeks.
    Sodium    perchlorate       (0.1 mol/liter): Dissolve 12.25
g sodium perchlorate          in de-ionized water and dilute             a stable baseline, but less than 90% of SCN added to
to 1 liter with water. Add 1.0 ml of “Brij-35” surfac-                   serum was analytically              accounted for (Table 1). Pol-
tant (30 g/dl), mix and store in a dark bottle at 4 #{176}C;             lay et al. (13) showed that under physiological                     condi-
prepare every two weeks.                                                 tions serum thiocyanate              is 40% protein bound, exclu-
   Dialyzer    recipient:      To 1 liter of de-ionized water            sively to albumin.            As shown in Table 1, use of stan-
add 1.0 ml of Brij-35 (30 g/dl) and mix; prepare as                      dards prepared in albumin solution (3 g/dl) and run
needed.                                                                  with a NaCl (0.15 mol/liter) diluent produced a high-
                                                                         er relative recovery. However, this recovery was still
Method Development                                                       less than 100% and required using inconvenient                       albu-
    The determination         is run on the AutoAnalyzer              II mm base standards.              In an attempt to displace SCN
 (Technicon      Instruments        Corp., Tarrytown,          N. Y.     from the serum albumin, NaC1O4 was used in place of
 10591) at 50/h with a sample-to-wash                 ratio of 2:1.      NaCl in the diluent.                 It can be postulated             that
Figure 1 shows the flow diagram. The 460 nm trans-                       NaC1O4, a chaotropic                 agent, which partially             de-
mission maximum            of the colorimeter         filter is the      natures the albumin, eliminates the binding sites for
same as the absorbance           maximum for the ferric thio-            the thiocyanate.
cyanate complex. Double dialyzers and mixing coils                          As indicated in Table 1, recovery was quantitative
were used to gain adequate sensitivity.                                  with three different concentrations                  of NaC1O4; a 30
    In our original experiments,           water was used to di-         mol/liter     NaC1O4 diluent caused a baseline shift simi-
lute the samples before dialysis; however, under these                   lar to that observed with water. We chose the 0.1
conditions the baseline shifted significantly   between                                         mol/liter   concentration   because      there   was   no appar-

aqueous standards     and serum specimens.    Substitu-                                         ent advantage in using higher concentrations.
tion of saline (0.10 or 0.15 mol of NaC1 per liter) gave                                           The reaction between ferric ions and thiocyanate
                                                                                                must take place in acid media because of the ease of
                                                                                                hydrolysis of ferric ions to ferric hydroxide. Karsten
                                                                                                     )
                                                                                                (14 found that the color of the ferric thiocyanate
                                                       CONNECTOR     ASSEMBLY   a.*177-BQ4-O2
                                                                                                complex is practically independent       of the concentra-
                                                                              b.*177-B004-O1    tion of nitric or hydrochloric   acid in strong solutions,
       WASTE                                   .                      FLOW RATE
               2x   #177-BO1O-O1     UPPER           TURN COIL                                  and he preferred nitric acid, which gave a more stable
               2    *177807801       LOWERI        *170-0103
                                                                                                color. Consequently     we chose to use a 1.5 mol/liter
                                                                                                HNO3 stock reagent, giving a final concentration           of
                                                                                                about 1 mol of HNO3 per liter.
                                                                                                    With Fe(N03)3 . 9H2O concentrations         of 2, 5, 10,
                                                                                                20, 50, and 100 g/liter, the recoveries of SCN added
                                                                                                to a serum pool were 100, 100, 100, 98.5, 95.5, and
                                                                                                92.0%, respectively.    Absorbance    and SCN       concen-
                                                                                                tration were linearly related in each case, but ade-
                                                                                                quate recovery was found only with the lower concen-
                                                                                                trations of ferric nitrate. Adequate sensitivity and a
                                                                                                low signal-to-noise   ratio became a problem with the
                                                                                                lowest ferric nitrate concentrations,    and we ultimate-
Fig. 1 Flow diagram of the analytical system used to deter-
         .                                                                                      ly selected 10 g of Fe(NO3)3 - 9H2O per liter as the
mine     thiocyanate               in serum                                                     optimum concentration.

                                                                                                            CLINICALCHEMISTRY.        Vol. 20, No. 10, 1974   1345
                                                                            AotaoJs STNftOS   I,oFt,I                                                              SWCN1
                                                                                                                                                            AGIJEOUS                    II”OI/IIWU




Fig. 2. Recording illustrating                                                                                                       I                      I

precision, stability, recovery,
carryover, and linearity of the
System




               250

                                                                                                        Table 2. Precision of the Method
                                                                                                                                                   Within-day                     Between-day
                                                                                       Control sample                              Mean           SD        CV, %                 SD          CV, %
               200                                   I
                                                                                    Pooled human
                                                                                       sera                                        98.6          0.83       0.84                 0.81         0.82
                                                     I
                                                                                    HylandAbnormal                                  69.2         1.26       1.83                 0.95         1.37
                                                                                    Hyland Normal                                  136.3         2.14       1.57                 1.54         1.12
               150
           0

          z
          0
                                                     I
          I                         I
                                                    V
                                                                                                        250-




                       U                             I
                                                                                                                                                                                         x




                      H
                                                                    I                                   200
                50




                                                                                                   -    150
                  0
                       NONSMOKERS                        SMOKERS                                  a                                       I
                      MALE       FEMALE        CIGARETTE       CIGAR                              z
           NUMBER      79          88              30               8
           MEAN        42          46              144             90
                                                                                                                                          S
           S.D.        15          18               48             59                                   100                                        x

Fig. 3. Serum thiocyanate concentrations in 205 healthy mdi-
viduals                                                                                                                                            x
No. subjects is gIven; mean and SD are in tmoIes   of SCNper        lIter                                                                x
                                                                                                                              x
                                                                                                         50-              x


   Under     the optimized   conditions,    recoveries of
                                                                                                                                                                    .   - CIGARETTES
100-101% were obtained for 50, 100, and 150 mol of                                                                                                                  x-   CIGARS
                                                                                                          0
SCN per liter added to a serum pool with an initial                                                            0
                                                                                                                   ---,

                                                                                                                              .5
                                                                                                                                          ,

                                                                                                                                         1.0
                                                                                                                                                    ,

                                                                                                                                                   1.5      2.0
                                                                                                                                                                ,            ,

                                                                                                                                                                           2.5          3.0
SCN      content of 60 imol/liter.    Table 2 shows the                                                                            PACKS CIGARETTES/DAY
                                                                                                               0              1          2          3           4           5           >5
precision of the method as assessed by analysis of                                                                                             CIGARS/DAY
variance of three different control samples, each run                              Fig. 4. Relationship between serum thiocyanate concentration
in duplicate    for 20 days. Carryover was calculated                              and no. cigarettes or cigars smoked per day (20 cigarettes
(15) to be 0.4% low-to-high    and high-to-low between                              per pack)
standards      of 20 mol    and 250 tmol of SCN        per liter.
Figure    2 illustrates  the in-run  precision, stability,     re-                 guishes individuals                             who are smoking cigarettes  from
covery,carryover,            and linearity    of the method.                       those who are not.                              Figure 3 summarizes  our results
                                                                                   for 205 normal individuals.  These subjects were not
Discussion                                                                         under a doctor’s care and were determined         to be
   The best way to judge whether this method fulfills                              healthy on the basis of a health history, physical ex-
its purpose  is, of course, to see whether it distin-                              amination, and a battery of chemical and hematolog-

1346      CLINICALCHEMISTRY,            Vol. 20, No. 10, 1974
                                    Table       3.   Weekly Serua SQI      concentxauona       in a Series
                                                        of 59 Healthy    Individuals


Subject     Ag.   Sex    ryp’                            Serna        conc   (,iijnol/liter)                            Mean            SD       CV, I
                         ske           uency                     during week no.                                       (.il/liter)
                                                            1       2      3            4        5     6

       1     23    F        N                              43     53        51         42                                  47           5.6      11.8
       2*    66    N        N                              36     51         41        37       50                         43           7.9      18.4
       3*    61    F        C               1             158    145       134       129       144                       142           10.0        7.0
       4     32    II       N                              67     66         59        54       54                          60          6.3       10.5
       S     45    II       N                              22     29         28        38                                   29          6.6       22.6
       6     49    P        N                              42     74         67        82                                   66         17.3       26.1
       7     43    N        N                              35     43         32        35                                   36          4.7       13.0
       8     49    V        N                              37     25         28        35       44                          34          7.5       22.3
       9     19    K        N                              31     33         36        31                                   33          2.4        7.3
   10        51    N        N                              51     47         73        89                                   65         19.6       30.3
   11        34    P        N                              32     48         42        55                                   44          9.7       22.0
   12        49    N        N                              94     91         96                                             94          2.5        2.7
   13        59    14       NQ                             79     81         72      101                                    83         12.4       14.9
   14a             N        N                              64     77         81       80        87                          78          8.5       11.0
   is        51    F        N                              62     67         71       70                                    68          4.0        6.0
   16        48    II       eR              3              81      73        73       71        72                          74          4.0        5.4
   17        45    N        N                              31      29        27       33                                    30          2.6        8.6
   1$        49    14       N                              95      63        65       76        78                          75         12.8       16.9
   19*       49    F        C               1             149    140       150       150       138                       145            5.9        4.1
   20        43    N        N                              34      30        26        23       55                          34         127        37.7
   2l        34    F        C               1.5           140    146       147       131                                 141            7.3        5.2
   22a       41    K        N                              54      50        42        44       35                          45          7.3       16.3
   23        40    P        N                              58      42        50        57                                   52          7.4       14.3
   24        38    N        N                              58      72        65        78       60                          67          8.4       12.5
   25        36    F        C               1             272    214       253        246      252                       247           21.1        8.5
   26        47    II       N                              62      74        62        61                                   65          6.2        9.6
   27”       25    F        N                              33      65        76        56                                   58         18.2       31.8
   28        31    14       N                              19      37        4].       29                                   32          8.1       25.7
   29        30    F        N                              43      39        47        42                                   43          3.3        7.7
   30        64    N        N                              15     11         16        16                                   15          2.4       16.4
   31        34    N        C               1             214    206       193       190       210                        203          10.6        5.2
   32a       29    F        c               1             102     97       124        109       99                        106          10.9       10.3
   33a       31    N        N                              50     45        43         46       36                         44           5.1       11.7
   34        39    F        N                              34      57       42         33       50                         43           9.2       21.4
   35        50    N        R               10            213    163       232        255                                 216          39.1       18.1
   36        26    II       N                              43      68        68        58       41                          56         13.1       23.5
   37        50    N        N                               27     27        28        35                                   29           5.9      20.2
   38        23    II       N                               38     43        52        53       53                          48           6.9      14.5
   39        43    II       c:              1               56     58        70        71       60                          63           7.0      11.1
   40        31    N        N                               64     63        59        44                                   58           9.3      16.1
   41        59    F        N                               35     29        43        41                                   37           6.3      17.1
   42        56    F        N                               44     41        35        33       38                          38           4.4      11.6
   43        54     II      N                               38     45        33        38       49                          41           6.3      15.6
   a         28     N       N                               67     45        45        50                                   52          10.4      20.2
   45a       26     F       N                               63     50        42        54       47                          51           7.9      15.5
   46        39     N           N                          39      25        26        35       40                         33            7.1      21.5
   47        27     N           NQ                         93      92        57        95      101         U3              90           11.5      19.4
   48        31     F           C           1.5           110     136       128       126                                 125           10.9       8.7
   49        31     F           N                          37      39        61        55                                    48         11.8      24.7
    50       33     N           N                          27      31        31        38                                    32          4.6      14.4
   51        55     K           N                          22      20        23        22       23                           22          1.2       5.6
   52a       26     F           N                          38      40        39        37       34                           38          2.3       6.1
   53a       28     N           N                          47      40        58        53       51                           50          6.8      13.6
   54        24     F           C           1             175     183       149       122                                 157           27.4      17.4
   55        55     F           C           1             222     203       203       190                                 204           13.2       5.4
   56a       36     N           N                          49      35        38        40       36                         40            5.6      14.1
   57a              F           N                          27      32        26        32       25                         28            3.9      13.6
   58        46     F           N                          32      30        29        28                                    30          1.7        5.8
   59        40     N           N                          47      45        41        42       47                           44          2.8        6.3




                                                a Conascutive       subjects with thie foothote are married to each other
                                                b         ,   Q-quit   re     than a year ago , C-cigarette    , cR-cigar
                                                C Nuaber    repreunta      packs  of cigarettes  or individual     cigars
                                                  usioked per day




                                                                                                CLINICAL        CHEMISTRY.           Vol. 20, No. 10, 1974   1347
                                                                            This work was supported           in part   by grant   No. 5R01    GM 17369-
ic tests. Each point in the Figure represents an aver-
age of determinations        on at least two specimens                 05 from the NIH, USPHS.
drawn from each subject at weekly intervals.           The             References
ability to discriminate    between cigarette smokers and               1. Delarue,       N. C., A study of smoking           withdrawal.      Can. J. Pub.
nonsmokers     is clear. With 85 tmol of SCN per liter                 Health       64, 55 (1973).
as a breakpoint,    only three false positives in 167 non-             2. Dastur,       D. K., Quadras, E. V., Wadia, N. H., et al., Effect of
smokers and two false negatives in 30 cigarette smok-                  vegetarianism        and   smoking      on vitamin B12,     thiocyanate, and fo-
                                                                       late    levels   in the    blood of    normal   subjects.   Brit. Med. J. 3, 260
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pipe smoker in the study had an average SCN con-                       8, 289 (1955).
                                                                       4. Densen,      P. M., Davidow,     B., Bass, H. E., and Jongs, E. W., A
centration    of 122 molfliter,      and one participant       who
                                                                       chemical    test for smoking exposure.      Arch. Environ. Health 14, 865
chewed tobacco had the lowest concentration                     ob-    (1967).
served among all subjects (12 jzmol per liter). There is               5. Wilson, J., and Matthews,          D. M., Metabolic interrelationships
a positive relationship          between the serum thiocy-             between cyanide, thiocyanate,          and vitamin B12 in smokers and
                                                                       nonsmokers.       Clin. Sci. 31, 1 (1966).
anate      concentration       and number           of cigarettes
                                                                       6. Pettigrew,   A. R., and Fell, G. S., Simplified colorimetric deter-
                          ,
smoked (Figure 4) but the correlation                is poor (r =      mination   of thiocyanate in biological fluids, and its application to
0.46). The correlation         between smoke inhaled and               the investigation of toxic amblyopia. Clin. Chem. 18, 996 (1972).
serum SCN          would probably be much higher. The                  7. Goldsmith, J. R., and Landsaw, S. A., Carbon monoxide             and
                                                                       human health. Science 162, 1352 (1968).
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                                                                       8. Astrup, P., Carbon monoxide and peripheral         arterial  disease.
may reflect inaccurate          estimates    of the iumber        of   Scand. J. Clin. Lab. Invest. 19, 193 (1967).
cigarettes smoked, the length to which the cigarette                   9. Ringold, A., Goldsmith,    G. R., Helwig, H. L., et al., Estimating
was smoked, the difference          between    filter and nonfil-      recent carbon monoxide exposures. Arch. Environ. Health 5, 308
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                                                                       10. Aldridge, W. N., A new method for the estimation of micro
SCN     concentrations          among    nonsmokers.
                                                                       quantities of cyanide and thiocyanate. Analyst  69, 262 (1944).
    Fifty-nine of the subjects had at least four consec-               1 1. Bowler, R. G., The determination     of thiocyanate     in blood
utive weekly blood samples drawn (Table 3). These                      serum. Biochem. J. 38, 385 (1944).
data show that the intraindividual        variation for ciga-          12. Van Peborgh, P. F., A continuous-flow      analysis system for
rette smokers        (CV, 8.8%) and nonsmokers           (CV,          thiocyanate (and cyanide)             in physiological preparations.      Biochem.
                                                                       Med. 6, 105 (1972).
16.0%) is significantly       less than the interindividual            13. Pollay, M., Stevens, A., and Davis,           C., Determinations    of
population     variation shown in Figure 3, where the                  plasma-thiocyanate         binding and the Donnan ratio under simulated
coefficient of variation for the cigarette smoker is                   physiological     conditions.    Anal. Biochem. 17, 192 (1966).
33% and for the nonsmokers            37%. It is also shown            14. Karsten,    P.,     Bdrage       tot   de colorimetrie, Thesis. Groningen
                                                                       (Holland),   1934.
that individual smokers have smaller relative ranges
                                                                       15. Recommended       scheme    for the evaluation            of instruments       for
than nonsmokers         because of the much higher mean                automatic  analysis  in the clinical biochemistry             laboratory.    J. Clin.
values.                                                                Pathol. 22, 278 (1969).




1348   CLINICALCHEMISTRY.            Vol. 20, No. 10, 1974

				
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