Polarographic Method for Rapid Microdetermination of Cholesterol

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					CLIN.      CHEM.       22/3,     3.36-340(1976)

Polarographic                                          Method                       for Rapid Microdetermination                                                                                    of

Cholesterol                                  with Cholesterol                                                   Esterase                  and Cholesterol                                                         Oxidase

Akio           Noma            and Keiko            Nakayama

Cholesterol     concentrations         in serum     are enzymatically                                               routine              assays,              hydrogen              peroxide               resulting                   from          the
determined      rapidly     by use of a polarographic             oxygen                                            catalytic               action              of      cholesterol                oxidase                  is     measured
analyzer    with a circuit     modified     to record    simultaneous-                                              colorimetrically                           by the coupled    systems   involving    ox-
ly the amount         and rate of oxygen           consumption.        The                                           idation            of either               methanol   in the presence    of catalase
final          assay          system,      assessed              from         the       oxygen              con-     (EC             or       phenol           and      4-aminoantipyrine                                    in the
sumption      value that we found to be optimum,                     consists
                                                                                                                    presence                of       peroxidase                   (EC                   These               reac-
of 1 ml of sodium         phosphate        buffer      (0.6 mol/liter,        pH
                                                                                                                    tions,         however,                   have       many          disadvantages:                            lengthy              re-
7.0) containing     NaN3 (10 mg/liter),           Triton X-100         surfac-
tant (10 mI/liter),    0.4 U of cholesterol              ester hydrolase,                                           action          time           and interfering                       substances                   (most              notably
and 0.6 U of cholesterol          oxidase.      Oxygen        consumption                                           bilirubin              and        ascorbic             acid).

and cholesterol     concentration        are linearly        related    to 8.0                                           This           paper            presents            a new           polarographic                             microde-
g/liter,  and only 10 tl of serum is required.           Replicate                                                  termination                      of serum              cholesterol                   with         use         of      two        en-
analyses    of pooled    serum   by the present     method     dem-                                                 zymes,              based            on     the     method             for      serum             glucose                deter-
onstrated     the following    inter-run  precision:     mean      =                                                mination                (5-7).
 1731   mg/liter,     SD     22.3 mg/liter,      CV                                     1.3%.     Biliru-
bin and ascorbic          acid    were      without                                  effect     on the              Materials                   and Methods
present       method,    unlike    the    enzymatic                                      colorimetric               Reagents
                                                                                                                         All        enzyme                    preparations                  were            from                 Boehringer
AddItional             Keyphrases:            pediatric        chemistry               #{149} intermethod            Mannheim                    Yamanouchi                      K. K.,           2-9,     Kandatacho,                             Chi-
comparison                                                                                                          yoda-ku,               Tokyo,               and      were used    without                           further        pun-
                                                                                                                     fication.            Cholesterol                    oxidase   (EC;                            cholesterol:
       Because              of clinical        importance                 and        analytical              dif-
                                                                                                                    oxygen   oxidoreductase)                                 from    Nocardia                           erythropolis
ficulties,           determination                  of serum         cholesterol                  has     been
                                                                                                                    had a specific    activity                            of approximately                             20 kU/g       or 20
the subject                   of numerous              investigations.                  Colorimetric
                                                                                                                    kU/liter.               Cholesterol                    esterase                (EC  ;                sterol-
reactions                  for cholesterol               determination                     have      been
                                                                                                                    ester        hydrolase),                    with      a specific              activity             of 10 kU/g                     or
plagued              with       a long       list      of difficulties:               lack        of speci-
                                                                                                                    20      kU/liter,             was          from       a microorganism                             (not             specified
ficity,          difficulty         in standardization,                       variable            reactivi-
                                                                                                                    by    the supplier).                          Glucose      oxidase                     (EC;     f3-D-
ty        of     esters,        unstable            and       corrosive              reagents,               and
                                                                                                                    glucose:oxygen                            oxidoreductase),                           210   kU/g,      was     of
many            interfering          substances.              Recently,              the      specificity
                                                                                                                    fungal      origin,     and    mutarotase                                     (EC;                     aldose-1-
provided               by       enzyme            reagents         has        been           introduced
                                                                                                                    epimerase),         with    approximately                                       6000 kU/g                      or 30 000
into           cholesterol           determinations                  (1-4).         In the               nzy-
                                                                                                                    kU/liter,      was from hog kidney.
matic            cholesterol-measuring                        systems            adopted                in the’
                                                                                                                         Sodium                 azide,          a strong              inhibitor                 for    catalase,                   was
                                                                                                                    dissolved              in buffer                  to give         a final       concentration                            in the
   Division          of Clinical    Biochemistry,             Tokyo    Metropolitan                 Geriatric
Hospital,          35-2, Sakae-cho,        Itabashi,         Tokyo,   Japan.                                        assay  system      of 10 mg/liter.   Sodium     taurocholate                                                                   and
   Received           Nov.    25, 1975; accepted             Dec. 24, 1975.                                         sodium     cholate    were purchased       from   Calbiochem,                                                                    La

336            CLINICAL        CHEMISTRY,           Vol. 22, No. 3, 1976
                                                                                                                                      action              of      any            catalase                      present,                         we        added                  NaN3,                    a
                                                                                                                                      strong             catalase                inhibitor,                      to the               reaction                         mixture.
                                                                                                                                             Determination                                 of true              cholesterol                           concentration                                    of
                                                                                                                                      standard       solution.                                  In the   reaction                                       cell was placed
                                                                                                                                      buffer     (see below)                                  containing    NaN3                                      (final  concentra-
                                                                                                                                      tion,    10 mg/liter),                                   50 .d of glucose                                       oxidase    solution
                                1 mm    HOLE                                                                                          (3150             kU/liter),                      40         .tl of        mutarotase                       solution         (30 000
                                                                                           OXYGEN         ELECTRODE
                                                                                                                                      kU/liter)                  and           30                 of cholesterol                            oxidase       solution      (20
                                                                                                                                      kU/liter).                  After                 setting               the         meter                  reading                     of the             oxy-
                                                                                                                                      gen        analyzer                  at a proper                          position,                        while                 continuously
                                                                                                                                      stirring              with               a stirring                      bar          covered                       with               Teflon,                we
                                                                                                                                      added   10     of D-glucose                                                        solution                    (2         mmol/liter)                            to
                                                                                                                                      give an oxygen    consumption                                                           value                  (A) corresponding
                                                                                                                                      to        total            D-glucose                          (a-anomer                           +         f3-anomer)                              in      the
                                                                                                                                      sample                solution.                        Subsequently,                                  10        zl         of           cholesterol
Fig.     1.   Reaction          cell (1-mI glass cell)
                                                                                                                                      standard                   solution                     was         added               to give                  a further                       oxygen
                                                                                                                                      consumption                              value      (B).                       Then,                 the            true                cholesterol
                                                                                                                                      concentration                              (mmol/liter)                               of standard                                 solution                  was
                                                                                                                                      calculated                   as 2             X      (B/A).
Jolla,         Calif.      92037          and          used          without               further              purifica-
                                                                                                                                               Measurement                                of free               and           esterified                           cholesterol                           in
tion.          Isooctyl          phenoxypolyethoxyethanol                                               (Triton             X-        serum.                In           the            reaction                     cell          was               placed                   phosphate
100)          and fatty           acid-poor      bovine   serum                                     albumin               were
                                                                                                                                      buffer             (0.6        mol/liter,                      pH         7.0,          and           containing,                             per         liter,
obtained                from       Sigma               Chemical                    Co.,      St.         Louis,            Mo.        10        mg        of      NaN3                    and            10         ml        of       Triton                      X-100).                     After
63178.   Polyoxyethylene            sorbitan      monooleate       (Tween
                                                                                                                                      adding                30       l          of        cholesterol                         oxidase                        and             setting                the
80), was from        Kanto     Chemical        Co., Inc., Tokyo.       Cho-
                                                                                                                                      meter              reading                 of the                  apparatus                          at        a proper                      position
lesterol    standard       solution        (“Preciset      Cholesterol”)
                                                                                                                                      while              continuously                                stirring                  the               mixture,                           10 zl                of
was a gift from   Boehringer                                        Mannheim      Yamanouchi
                                                                                                                                      serum              sample                 was               added              to give                an oxygen                           consump-
K. K., Tokyo.   All other                                       chemicals    used  were    of                                   re-
                                                                                                                                      tion         corresponding                                   to the             free          cholesterol                              in the             sam-
agent          grade.
                                                                                                                                      ple.        Then            20 ul of cholesterol                                             ester             hydrolase                         (20        kU/
Apparatus                                                                                                                             liter) was added                                       to     give             a further                       oxygen                     consump-
                                                                                                                                      tion corresponding                                           to the             esterified                      cholesterol                              in the
       To      measure               oxygen           consumption,                           we          used      a po-
                                                                                                                                      sample               (Figure                   2).
larographic                    oxygen            analyzer       with                      oxygen            electrode
 (used         in all     models               of the           Glucose Analyzer,                              Beckman                Results
Instruments                    Inc.,     Fullerton,                 Calif. 92634)                        and record-                  Analytical                     Variables
er. A 1.0-ml                drumshaped                          glass       cell     with           small             Teflon-
coated           stir     bar        (Figure               1) was           placed            in the              thermo-                      Optimal                 pH.              The         optimal                   pH            for       assay                  system               was

statically              controlled                system.                With         this          apparatus                   we    studied                  by determining                                   the           maximal                         rate            for       choles-
                                                                                                                                      terol             standard                     solution.                       A broad                         pH            optimum,                         be-
could          monitor            and          simultaneously                        record              the      amount
and rate                of oxygen   consumption.                                    We        used             microsyr-              tween               6.5-8.0, was observed                                                    (Figure                    3).            Subsequent

inges-lO,                 25, 50, and 100 id-to                                     add       serum              samples              studies              were made   at pH 7.0.
                                                                                                                                               Buffer             concentration.                                     Sodium                      phosphate,                            pH          7.0,
 and        reagents            to the         reaction                 mixture.
                                                                                                                                      was         used           as the                 buffer             at final                 concentrations                                     ranging
       This       method             is based                  on the        absolutely                  specific               ox-
 idation          of free         cholesterol,                      which           is formed                  with        cho-
 lesterol         esterase             (CE),          with          cholesterol                oxidase                  (CO).
 cholesterol              ester         + H2O              -             cholesterol                 +

                                                                                              fatty            acid             (1)                                                                                                                                                            1.
                                                                                                                                                                                                                cnoLaTz*o(.                      $YDROIASE         -
 cholesterol               + 02                     4-cholesten-3-one                                +

                                                                                                            H2O2                (2)                                                                                                  CS.UTII.                GX1DA1

 If catalase             is present                in the           sample,            the          hydrogen                per-
 oxide         formed            in equation                     2 is decomposed                          to 0.5           mole       Fig.        2.      Determination                             of        free          and         esterified                       cholesterol                        in
 of oxygen    and 1 mole of water,  and the oxygen    thus                                                                            serum
                                                                                                                                       Values       F, E, and S are the amounts of oxygen consumed                                                                           corresponding                  to
 formed    may be again used for the oxidation  of anoth-                                                                              free      and esterifled cholesterol in the sample. and cholesterol                                                                      standard            solu-
 en molecule                of cholesterol                       by equation                   2. To            block           the    tion.     respectively.             Incubation               was        at 37 #{176}C

                                                                                                                                                                                CLINICAL                  CHEMISTRY,                        Vol.          22,          No.    3, 1976                  337
                                                                                                                                                                                                                                                                                                      i.e     unit
                                      /CH0LESTER0L                                                               300 mg/dO                                             70

                                                                                                                                                                                                                                                                                                      1.4     unit
                S                                                                                                                                                      sol.
                                                                                                                                                                                                                                                                                                      1.0     unit
                                                                                           CHOLESTEROL           200   mg/dO

                                                                                                                                                                                                                                                                                                      0.6     unit



                                                                                                                                                                                                                                                                                                      0.2     unit
                                                                                           CHOLESTEROL           100   mg/dO


                                                                                                                                                                                                                           100                   200                      MS                    400
                                                                                                                                                                                                                           CHOLESTEROL CONWITRATION                          (ss/100        ML)

                                               6.0                 7.0                  8.0
                                                                                                                                             Fig. 5. Effect of amount                                                             of cholesterol                            oxidase                   on the rate of
                                                                                                                                             oxygen consumption
Fig. 3. Effect                of pH on the reaction                               rate of oxygen                          consump-
0.6 mol/Ilter           sodium        phosphate           buffer         used.     Here,       and in Figures                  4, 5, 7, 8.

                                                                                                                                                                                                          .--..---...--.                   .                                     3OVINE     ALWJMIN

values      on ordinate          are oxygen            consumption               rate                                                                                             0                                                                                                              0

                                                                                                                                                                                  I                                                                                            TRITON       X100

                          ::                         /                                                      \#{149}
                                                                                                                                                               un      80


                                                                                                                                                                                                .--....                      I’
                                                                                                                                                                                                                                    /.0..              ...



                                           #{149}/                                                                                                                     40


                                                                                                                                                                              0                               0.2                    0.4                      0.6          0.8                  1.0

                                                                                                                                                                                                                                  DETERGENT CONCENTRATION

                                  0              0.2               (1.4             fl.6                 fl.R            1(1
                                                                                                                                             Fig. 6. Effects                   of various                                           concentrations                                  of detergents                       on the
                                                                                                                                             reaction          rate by using cholesterol                                                                       standard                   solution
Fig. 4. Effect                of phosphate                    concentration                       on the rate of oxy-
gen      consumption

                                                                                                                                             ethoxydodecane                                                   (an                   unspecified                                  concentration)                                   as
                                                                                                                                             detergent,                    was             used,                                 effects                of various                         concentrations
from         0.1        to 1.0         mol/liter,                  in 0.1           mol/liter                    increments.                 of other              detergents                                              on        the          rate               of      cholesterol                             oxidase
A range              of 0.6-0.7                 mol/liter                  was          found              to be optimal                     reaction               were              as shown                                            in Figure                        6. Bovine                          serum              al-
(Figure             4).                                                                                                                      bumin             slightly                     activated                                           the            reaction,                    in the                   concen-
   Cholesterol        oxidase                               concentration.                                 We measured                       trations              used           in this                                    experiment.                                  Triton                  X-100                slight-
oxygen      consumption                                  and reaction                         rate          of the oxida-                    ly inhibited    the activity                                                                        of cholesterol                                      oxidase.               An-
tion     of free cholesterol            with      various      amounts     of cho-                                                           other non-ionic    detergent,                                                                        Tween                     80, significantly         in-
lesterol       oxidase     at various           concentrations         of choles-                                                            hibited            the           reaction                                            rate.           On the                     contrary,        maximal
terol     standard       solution.        As shown           in Figure    5, reac-                                                           inhibitions                    were                           observed    when       bile                                                    salts     (1 or 2 g/
tion     rate    was linear        with      respect       to enzyme      concen-                                                            liter)       were             present,                            but the activities                                                         increased      again
tration       when     more      than      1.8 U of cholesterol           oxidase                                                            as bile           salt          concentrations                                                           were              increased                           above           3 g/
was        present.             On       the         other       hand,     oxygen  consumption                                               liter.
was        linear        with as little                      as 0.2 U of cholesterol       oxidase                                                 In    contrast                     with                             serum                     as           the         sample,                     these            deter-
present.     In            subsequent                         measurements        of cholesterol                                             gents         had             different                                        effects.                    As          shown                   in Figure                     7, in
concentration,                        we        used          oxygen                    consumption                            as   our      the        case          of      buffer                                       without                        detergent                          no             activity              of
measure.                                                                                                                                     cholesterol                     oxidase                                        was                recorded.                       On           adding                    Triton
      Effects            of various                    detergents.                         When                 10 l           of cho-       x-100             (final             concentration,                                                               1 ml/liter),                           the            activity
lesterol             standard                  solution,                  containing                        hydroxypoly-                     was        sharply               increased                                             and           reached                           the         maximal                   rate;

338         CLINICAL            CHEMISTRY.                   Vol. 22, No. 3, 1976
                        15                                                                                                                                                           with              10-20                .sl (0.2-0.4                        U)         of        cholesterol                           esterase.
                                                                                                                                                                                     Unexpectedly,                                greater                 amounts                    of the              enzyme                     inhib-
                                                                                                                                     TRITON      0-100

                                                                                                                                                                                     ited         the          activities.                    Thus,             20 tl            of cholesterol                               esterase
                                                                                                                                                                                     was         used           in subsequent                              studies.
                                                                                            ,S_         _      _      _
                                                                                                                                                                                            Sensitivity.                          It    is apparent                        from             the         results                of dilu-

                                                                                        I                                      ‘ ‘    O00UM      T0000CHOLAT!

                                                                                                                                                                                     tion         experiments                               that,  with   a 10-j.tl sample,      a choles-
                                                                                                                                                                                     terol         concentration                               of 125 mg/liter     is measurable       with
                                                                                                                                                                                     the      present                  method.
                            5                                                                                                                                                               Linearity.                   The standard                                 curve                prepared                      with            van-
                                                                                                                                                                                     ous          concentrations                                of         cholesterol      standard                                          solution
                                                                                                                                                                                     was          a straight                      line         up          to 8.00 g/liter.       Another                                          curve

                                       /               I’
                                                                                                                                                                                     prepared                        with              various                  volumes                      of         serum                   sample
                                 #{149}--m--...--                  ......#{149}....                    .......-...........
                                                                                                                                      BOVINE     ALBUMIN
                                                                                                                                                                                     (total             cholesterol,                         1540           mg/liter)                      was          linear             up            to 40
                                 0                          0.2                   0.4                     0.6                      0.8           1.0                                 1ul of serum,                      corresponding                                to a cholesterol                                concentra-
                                                                         DETERGENT CONCENTRATION (2)                                                                                 tion         of about                   6000            mg/liter.
                                                                                                                                                                                            Reproducibility.                                    The               precision                        of          the             present
Fig. 7. Effects         of various concentrations of detergents                                                                                                         on the
reaction         rate by using serum as a sample                                                                                                                                     method                    was           determined                         by         performing                              12      replicate
                                                                                                                                                                                     assays              of pooled                     serum.               The            mean              was          1731             mg/liter,
                                                                                                                                                                                     with          a standard                          deviation                   of 22.3                 mg/liter                 and             coeffi-
                                                                                                                                                                                     cient             of variation                of 1.29%.                           The                 day-to-day                       CV was
                                                                                                                                                                                     1.95%,               mean                1696 mg/liter,                            and                 standard                      deviation
                                                                                                                                                                                     33.1         mg/liter.

                                                                                                                                                                                     Correlation                        with            Results                 by Other                     Methods
                            20                                                                                                     CHOLESTTHOL           330    mg/dl
                                                                                                                                                                                            Thirty-two                        samples                  were            assayed                    by using                    the          Au-
                                                                                                                                                                                     toAnalyzer                        II,        with          isopropanol                            extraction,                         and            also
                            15                                                                                                                                                       by          the          method                    of      the          Boehninger                            enzymatic                             color
                                                                                                                                                                                     test.         Both              procedures                       were            those                 of the             manufactur-
                                                                                                                                                                                     er.         The          resulting                     equations                  are:           y       (AutoAnalyzer)                                      =
                            10                                                                                                     CHOLESTEROL           145    mg/dl
                                                                                                                                                                                     0.793             x + 17.72                       (r           0.94)            and         y     (enzymatic)                              =        1.037
                                                                                                                                                                                     x       +     16.15              (r      =        0.985).              Clearly,                  there           is good                       agree-
                                                                                                                                                                                     ment               between                    the         present                 method                      and    those                       com-
                                                                                                                                                                                     pared              with          it.

                                                                                                                                                                                     Effect              of Interfering                             Substances
                                                     0.2           0.4                0.6                    0.8             1.0          1.2
                                                                  CHOI.ESTEROI            (ussi)
                                                                                                                                                                                             A native                   human                   serum                was             mixed               with              solutions
                                                                                                                                                                                     containing       either      bilirubin                                           or        ascorbic                  acid             and            ana-
Fig. 8. Effect                  of amount                                    of cholesterol                                        ester         hydrolase               on the
                                                                                                                                                                                     lyzed.     These     substances                                            in concentrations                                        up         to      100
rate of oxygen                        consumption                                           with serum samples                                                   of low and
high cholesterol                         concentrations                                                                                                                              mg/liter                   were without      effect                                   on either                     the            amount                   or
0.6      mol/liter      sodium                  phosphate                             buffer.                  pH 7.0.                containing                1 ml of Triton       the         rate          of oxygen    consumption.
x-loo       per lIter

                                                                                                                                                                                             Since             Kadish                  and          Hall        (8)        reported                     the         determina-
 no further                     change                             was observed                                                    with          up to 10 ml of                      tion          of D-glucose                              with          an        oxygen                 electrode                      and            /3-D-
 Triton              X-100                 per                liter.                  The                     activity                        increased                 gradu-       glucose                   oxidase                  (EC                   in     1965,              blood                 glucose
 ally       with            increasing      concentrations          of                                                                                   bile salt    and            has           been              determined                             with            similar                     techniques.                           At
Tween                80.        No activity       was      observed                                                                                       with    bovine             present,                  glucose                 in serum,                   plasma,                   and         urine                is deter-
serum            albumin                               present                              in any                           of the              concentrations                      mined                in         the          routine                  assay            by         the         glucose                      oxidase
tested.           The            rate of the                                          coupled      reaction                                               with   choles-             method     by use of a polarographic                                                                analyzer                       with a             cm-
 terol         ester             hydrolase                                            and    cholesterol                                                 oxidase      also           cuit modified    to record     the rate                                                           of oxygen                        consump-
 reached              the        maximal                                     value                     with                   1 ml of Triton                            X-100        tion              (9).          However,                       Okuda                  et        al.         (5-7)              reported                          a
 per     liter.   This       concentration             of Triton       X-100      was                                                                                                method                    for      determining                             glucose                from              the            amount                    of
 used      in subsequent          studies.                                                                                                                                           oxygen                   consumed.                        As         shown                 in    Figure                  5,        cholesterol
    Cholesterol          esterase        concentration.          Under      optimal                                                                                                  determination                       from                       the     rate            of oxygen        consumption
 conditions          of pH,         buffer       concentration,          detergent                                                                                                   requires                   a substantial                             amount              of cholesterol         oxidase
 concentration,                                 and              cholesterol      oxidase                                                                amount,              the    even    if                only    free                    cholesterol                        is being                         determined.
 effects             of various                               amounts        of cholesterol                                                                esterase             on   Moreover,                     the rate                    of oxygen                        consumption                            varies                ac-
 the    reaction       rate were studied,      with                                                                                                 use of serum                     cording               to the amount                                of oxygen                          contained                      in the re-
 samples        of high and low concentrations                                                                                                        of cholester-                  action              cell. Thus, our                              method     for                       cholesterol                      determi-
 ol.     As seen                 in Figure                                     8, reaction                                         rates           were             maximal          nation                   is based                 on       the         amount                    of oxygen                          consumed

                                                                                                                                                                                                                              CLINICAL                CHEMISTRY,                      Vol.        22,     No. 3, 1976                        339
by free cholesterol    catalyzed     by cholesterol    oxidase    to                                                                We thank             Messrs.           S. Matsuzaki           and       Y. Hikawa,              Beckman-Tosh-
                                                                                                                              iba      Ltd.,     for    their      cooperation.
4-cholesten-3-one.      Measurement           of the amount       of
oxygen     consumed    is influenced        by the atmospheric
oxygen,     but in the present      method,     with the specific
cell, diffusion                of atmospheric                          oxygen             into the test so-                   1. Richmond,                   W.,    Preparation             and      properties            of a cholesterol           oxi-
                                                                                                                              dase     from     Nocardia               sp. and          its application              to the         enzymatic       assay
lution     was              scarcely     observed                         during              the measure-
                                                                                                                              of total     cholesterol               in serum.           Clin.  Chem.             19, 1350         (1973).
ment,          because              the      baselines               of the            chart         were            abso-    2. Allain,           C. C., Poon,              L. S., Chan,          C. S. G., et al., Enzymatic       deter-
lutely  stable.                                                                                                               mination            of total  serum              cholesterol.          Clin.  Chem.    20, 470 (1974).
    The   present                    method              for       determining                       cholesterol              3. Tarbutton,                  P. N., and     Gunter,               C. R.,       Enzymatic            determination
has       several           advantages:                  rapidity,               sensitivity,                  and no         of total         cholesterol        in serum.     Clin.             Chem.         20, 724        (1974).
                                                                                                                              4. Witte,    D. L., Barrett,                      D. A., II, and Wycoff,        D. A., Evaluation                             of
interference                 from           bilirubin             or       ascorbic              acid.          In     less
                                                                                                                              an enzymatic       procedure                        for determination         of serum     cholesterol
than          2 mm          total         cholesterol                can         be      determined                    in a   with the Abbott      ABA-100.                        Clin.  Chem.     20, 1282 (1974).
sample.              Free     and         esterified            cholesterol                    can    be succes-              5. Okuda,             J., and        Okuda,          G., A rapid            polarographic               microdetermi-
sively         determined               within            4     mm.          Moreover,                   the       mini-      nation           of glucose           with      glucose         oxidase.         Clin.       Chim.       Acta     23, 365
mum           detectable              amount             of cholesterol                     by the             present
                                                                                                                              6. Okuda,                J., and       Miwa,         I., Polarographic                   microdetermination                of
method               is about             1 g     (10            of 125               mg/liter           solution).           D-glucose            anomers            with        fl-D-glucose            oxidase.         Anal.       Biochem.         39,
The       cholesterol                in as little              as 1              of serum                or plasma            387 (1971).

can      be measured.                                                                                                         7. Okuda, J., and                     Okuda,          G., Rapid            and   successive              microdetermi-
                                                                                                                              nations           of oxygen,            D-glucose,          and     its      anomers        in        blood     with    fl-D-
                                                                                                                              glucose            oxidase           and     oxygen          electrode.            Biochem.               Med.       7, 257
      In conclusion,                      although             the present                     method                is not   (1973).
especially            suitable      for            routine             assay of many     samples,                             8.  Kadish,     A. H., and Hall,      D. A., A new method                                             for the continu-
it      has      special       research                 and            pediatric  applications.                               ous monitoring       of blood   glucose   by measurement                                             of dissolved    oxy-
                                                                                                                              gen. Clin.    Chem.    1 1, 869 (1965).
With           the      present             method              we         are        studying              lipopro-
                                                                                                                              9. Kadish,     A. H., Litle,    R. L., and Sternberg,          J. C., A new                                              and
teins         as substrate                 and     the         interaction                 between               deter-       rapid   method    for the determination        of glucose    by measurement                                                 of
gents         and       lipoproteins                or lipid            micelles.                                             rate ofoxygen      consumption.       Clin. Chem.     14, 116 (1968).

340           CLINICAL        CHEMISTRY,                Vol. 22, No. 3, 1976

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