are there and what is their clinical significance?
Clin Chim Acta
1981;115:3-8.
10. Sax SM, Moore JJ, Giegel JL, Welsh M. Atypical increase in
serum creatine kinase activity in hospital patients. Clin Chem
197622:87-91.
11. Wu AHB, Bowers GN. Evaluation and comparison of immunoinhibition and immunoprecipitation
methods for differentiating MB
from BB and macro forms of creatine kinase isoenzymes in patients
and healthy individuals. Clin Chem 1982;28:2017-21.
12. Oliver IT. A spectrophotometric
method for the determination
of creatine phosphokinase and myokinase. Biochem J 1955;61:11622.
13. Bohner J, Stein W, Steinliart B, Wurzburg U, Eggstein M.
Macro creatine kinases: results of isoenzyme electrophoresis and
differentiation of the immunoglobuhn-bound type by radioaasay.
Clin Chem 198228:618-23.
14. Wu AHB, Herson VC, Bowers GN. Macro creatine kunase types
1 and 2: clinical significance in neonates and children as compared
with adults. Cliii Chem 198329:201-4.
15. Massey TB, Goe MR. Transient creatine kinaae-BB activity in
serum or plasma after cardiac or respiratory arrest. Cliii Chem
1984;30:50-5.
16. Stein W, Bobner J, Steinhart R, Eggstein M. Macro creatune
kinase: determination and differentiation of two types by their
activation energies. Clin Cheni 198228:19-24.
17. Hayashi T, Tanaka T. Appearance of cathodic band in the
electrophoretogram of blood creatine kinase isoenzyme-MM fraction during hypoxia in rats Cliii Chem 1985;31:533-.6.
18. Prabhakaran V, Nealon DA, Henderson AR Interaction between human IgG and human creatine kinase isoenzyme-1 m
serum: a route for the intravascular catabolism of creatune kunase1? Clin Chem 197925:112-6.
19. Cuestas B.. Creatune kinase isoenzynies in high-risk infants
Pediatr Res 1980;14:935-8.
CLIN. CHEM. 32/4, 691-693 (1986)
Immunoradiometric
Assayof Thyrotropinas a “First-Line”Thyroid-Function
Test in the RoutineLaboratory
M. A. Hopton and J. S. Harrop
We compared the utility of a sensitive immunoradiometnc
assayfor serum thyrotropinas a “first-line”thyroid-function
testwith a strategy based on first measuringtotal thyroxinin
serum. The immunoradiometricassay appears to distinguish
primary hypothyroidism and hyperthyroidism from euthyroidism in “new” patients.The role of thistest in monitoringantithyroidtreatment or thyroxin-replacementtherapy is not yet
established, there being particular difficulty
in interpreting low
thyrotropinconcentrations In such patients. Nevertheless,
becausea normalthyrotropinconcentrationin most,if notall,
situationssignifiesthe euthyroidstate, thyrotropindetermination by immunoradiometricassay meritsconsiderationas an
Initialtest by laboratoriesperformingthyroid-functiontests.
AddItIonalKoyphras.s:
thyroid status
sening
of most RIAs for thyrotropin
(TSH,
hormone) precludes accurate measurement of TSH in all euthyroid subjects and in hyperthyroid
patients, whose TSH concentrations
are low.1 Given the
increased
sensitivity of imxnunoradiometric
assays (m)
for TSH (1,2), or of carefully optimized RIA techniques for it
(3,4), some have suggested that this single laboratory test
distinguishes hyperthyroid
and primary
hypothyroid states
from euthyroidism. Such reports have concentrated
mainly
on patients referred to specialist endocrine clinics. We have
compared,
in a routine laboratory setting, the Boots-Celltech TSH-mz
as a “first-line”
test of thyroid function
against our existing repertoire of thyroid tests.
The insensitivity
thyroid-stimulating
Department of Chemical Pathology, Derby City Hospital, Derby,
DE3 3MB, U.K.
‘Nonstandard abbreviations: TSH, thyrotropin; msA, immunoradiometric
assay, 1I’4 total thyroxun.
Received October 2, 1985; accepted January
21, 1986.
Patients and Methods
We studied 320 consecutive requests for thyroid assessment of patients in the following categories: (a) 238 “new”
patients, who were not receiving
treatment for thyroid
disease; (b) 57 patients receiving
thyroxin
for primary
hypothyroidism; (c) 25 patients being treated medically for
hyperthyroidism.
Together, these categories compose 91% of
the total thyroid workload.
Present test “stmteg”. Currently, we first measure
the
concentration of total thyroxin (IT4) in serum. If IT4 is <70
nmol/L, we then measure
TSH (by RIA); if TF4 is >120
nmol/L, we measure
thyroxin-binding
globulin (and when
indicated,
total triiodothyronine). This “strategy range” (5)
for ‘1’F. represents the mean ± 1.4 SD of the normal reference interval (see Figure 1).
Analytical
methods. Our existing set of thyroid tests
comprises radioimmunoassay
of TF4, total triiodothyronine,
and TSH in serum and measurement of thyroxin-binding
globulin by immunoelectrophoresis.
The between-batch
CV
over the working ranges of these assays is <8%.
For this study we also measured
serum TSH by mM
(“Sucrosep” TSH; Boots-Ceiltech
Diagnostics Ltd, Slough,
Berkshire
SL1 4ET, U.K.). In this two-site IR.,
two
monoclonal antibodies
are used, one labeled with 1251 and
the other covalently bound to a solid phase (Sephacryl 8300,
Pharmacia). Separation of bound from unbound labeled
monoclonal antibody involves passage through a sucrose
solution, the bound antibody settling closer to the bottom of
the tube. Assay sensitivity, derived from 20 replicate analyses of TSH-free serum, is typically <0.08 milli-int. unit/L
(95% confidence limits). Between-batch
CVs at mean concentrations of TSH of 0.55, 1.1, 11.0, and 28.1 milli-int.
unitsfL were 7.1%, 5.1%, 3.7%, and 6.2%, respectively (n =
20 each).
CLINICALCHEMISTRY,Vol.32, No.4, 1986
691
and TSH-UULA in 26 hyperthyroid
patients who had been
treated with antithyroid
drugs for periods ranging from a
week to longer than 20 months. There was no significant
difference in T’F4concentrations between those with normal
or low TSH-mit.& concentrations. Normal ‘FF4 concentrations were observed in 70% of those with reduced TSH-ix
concentrations.
200-
x
x
150-
Discussion
100-
#{149}
..#{149}.
#{149}
:..
..
U
50U
.
U
U....
<0.07
I
I
I
0.12-
0.51-
5.5
0.50
5.5
TSH, mull-mt. units/I
FIg.1. Total thyroxin(U4) andthyrotropin (TSH)concentrations
In238
consecutive
“new”patients(i.e.,notbeingtreatedforthyroiddisease)
OuWLiossdcate the reference intetval (mean ±250) for TT4, the kww lines
the “strategy” range (mean ±1.4 SD).
#{149},
thyroidstatusby TT4.based
leststrategy:euthytdd.
X,thyroid
statusby TT4-based
leststrategy:hypentiyrold.
U, thyroidstatus by 1T4-based test strategy: pdmwy hypothyroid.
Conversion:SI fo Vac9lonalunits:thy,oxln1 nmoVL 0.07 gf10O mL
Results
New patients. Of the 238 patients not receiving treatment,
those considered hypothyroid, hyperthyroid, or euthyroid by
our present strategy numbered 14, four, and 220, respectively. Results of the TSH-Im& identified the hypothyroid and
hyperthyroid
patients. Of the 220 euthyroid patients, 198
(90%) had TSH-maa
values within the normal reference
interval (0.51 to 5.5 milli-int. units/L). Of the remaining 22
patients, six had mildly increased TSH (5.8-7.7 milli-int.
units/L) and IZ >70 nmol/L; one of these patients, in whom
the serum ‘Fr4 concentration was 135 nmol/L, was taking
the oral contraceptive pill. Of the 16 patients with belownormal TSH-im& concentrations
(<0.5 milli-int. unitlL),
three had undetectable TSH, but normal triiodothyronine
concentrations.
TSH (RIA) concentrations were normal in
two patients with TT4 <70 nmol/L and TSH-m
between
0.12 and 5.0 milli-int. unitsfL.
Patients receiving thyroxin. The 57 patients receiving
thyroxin for primary hypothyroidism
showed a statistically
significant correlation
(r
-0.44; p <0.001) between TSHmtt and IZ concentrations in serum. However, the I’F4
concentration
could not be predicted from the TSH concentration; the proportions of patients with TF4 results within
the normal range who had low, normal, or high values for
TSH-m
were 24%, 28%, and 48%, respectively.
Patients being treated for hyperthyroidism.
There was no
correlation (r = -0.21; p >0.1) between TT4 concentration
692
CLINICALCHEMISTRY,Vol.32, No.4, 1986
For those “new” patients who are euthyroid by present
tests, diagnosis is secured by the single finding of a normal
TSH concentration by flth1A. Primary hypothyroidism
is
indicated by an increased TSH-mMA concentration. Use of
TSH-m
as a “first-line” test also identifies patients with
possible mild hypothyroidism
who have “normal” ‘FF4 with
above-normal
TSH. However, such results can be seen in
subclinical hypothyroidism, and the benefits of identifying
such patients may be uncertain (6, 7); defining the significance of this condition is not made any easier by the small
component contribution
of within-individual
variation to
the “normal range” for thyroid hormones (8).
As shown by Seth et al. (9), hyperthyroidism
is accompanied by undetectable TSH concentrations
in the BootsCelltech assay. However, a significant number of our apparently euthyroid patients had decreased TSH-mru
concentrations. Reported lower limits for this assay range from 0.3
to 0.5 milli-int. unit/L (10), and further work is required to
define reference intervals in the hospital population, where
drug effects and illness (11) may significantly
affect ThH
secretion. In patients considered hyperthyroid
or primary
hypothyroid
from a TSH-m
result, measurement of serum thyroid hormone concentrations would be indicated to
(a) confirm the diagnosis and (b) assess biochemical severity
of the disease before treatment.
While increased TSH-m
concentrations indicate suboptimal thyroxin replacement in treated hypothyroidism, enhanced intracellular de-iodination of thyroxin to triiodothyronine in the pituitary, as compared with other tissues, may
suggest that decreased TSH concentrations would not necessarily indicate overtreatment (12). Whether this seriously
compromises the use of sensitive TSH-m
methods as
“first-line” tests for monitoring replacement therapy remains to be seen.
The lack of correlation between ‘Fr4 and TSH-m
in
hyperthyroid patients who are undergoing treatment possibly relates to prolonged suppression of the pituitary thyrotroph (13). Whether the appearance of detectable serum
TSH by IRMA might predict therapeutic
success (14), or its
disappearance
subsequently predict relapse [as claimed for
TSH responsivity of thyroliberin (15)], again must await
further study.
The “Sucrosep” TSH-ntMA has proved to be a reliable and
robust method in our laboratory. Between-batch reproducibility and sensitivity of the method are both satisfactory
(16). Although use of TSH-IRMA as a “first-line” test simplifies laboratory work, the clinical acceptability
of replacing
the traditional
thyroxin measurement
by
requires further discussion and investigation. At first sight,
however, determination
of TSH by “Sucrosep” isa1t has
much to commend it in distinguishing
hyperthyroid
and
primary hypothyroid states from euthyroidism.
We thank Boots-Celitech Diagnostics Limited for kindly donating
reagents for this study.
References
for the thyrotrophun
1. Caldwell G, Kellet HA, Gow SM, et al. A new strategy for
thyroid function testing. Lancet 1985;i:1117-9.
2. Cobb WE, Lamberton RP, Jackson IMD. Use of a rapid, sensitive
1984891334-6.
immunoradiometric
assay for thyrotropin
to distinguish
normal
from hyperthyroid subjects. Clin Chem 1984;30:1558-60.
3. Wide L, Dahlberg PA. Quality requirements
of basal S-TSH
assays in predicting an S-TSH response to TRH. Scand J Cliii Lab
Invest 1980;40(suppl 155):101-10.
4. Evans M, Croxson MS, Wilson TM, Ibbertson HK. The screening
of patients with suspected thyrotoxicosis using a sensitive 1H
radioimmunoassay.
Clin Endocrunol 198522:445-51.
5. Britton KE, Quinn V, Brown BL, Ekins RP. A strategy for
thyroid function tests. Br Med J 1975;iii:350-2.
6. Ridgway EC, Cooper DS, Walker H, Rodbard D, Maloof F.
Peripheral responses to thyroid hormone before and after x-thyroxme therapy in patients with subclinical hypothyroidism.
J Cliii
Endocrinol Metab 1981;53:1238-42.
7. Bell GM, Todd WFA, Forfar JC, et al. End-organ responses to
thyroxine therapy in subclinical hypothyroidism.
Cliii Endocrunol
1985;22:83-9.
8. Harrop JS, Ashwell K, Hopton MR. Circannual
and withinindividual variation of thyroid function tests in normal subjects.
Ann Olin Biochem 1985;22:371-5.
9. Seth J, Kellett HA, Caldwell G, et al. A sensitive immunoradiometric assay for serum thyroid stimulating hormone: a replacement
releasing
hormone
test? Br Med J
10. Sucroeep’ TSH-a first line test in the assimpnt
of thyroid
dysfunction. In: Abbot SR. Daniel SG, eda. Proc. of the second BootsCeiltech Symposium in Thyroid Function. Slough, U.K.: BootsCelitech DiagnosticsLtd., 1985.
11. Wehmnnn RE, Gregerman RI, Burns WI!, Saral B, Santos GW.
Suppression of thyrotrcpin
in the low-thyroxine state of severe
nonthyroidal illness. N Engi J Med 1985;312:546-.62.
12. Larsen PR. Thyroid-pituitary
interaction. Feedback regulation
of thyrotropun secretion by thyroid hormones. N Engl J Med
1982;306:23-32.
13. hngbar SI!, Woeber KA. The thyroid gland. In: WilliAmA RH,
ad. Texthook of endocrinology, 6th ad. Pbiladelphia
WE Saunders
Co., 1981:117-247.
14. Fischer HRA, Hackeng WHL, Schopman W, Silberbuach J.
Analysis of factors in hyperthyroidism, which determine the duration of suppressive treatment befare recovery of thyroid stimulating
hormone secretion. Clin Endocrinol 1982;16:575-85.
15. Gardner DF, Utiger RD. The natural history of hyperthyroidism due to Graves’ disease in remission: sequential studies of
pituitary-thyroid regulation and various serum parameters. J Olin
Endocrinol Metab 1979;49:417-21.
16. Musto JD, Piraolsnte JM, Chesarone VP. A comment on
thyrotropin measurement and evaluation [Opinion] Olin Chem
1984;30:329-30.
CLIN.CHEM.32/4, 693-696 (1986)
Determinationof VanillylmandelicAcidwith Ion-PairChromatographyand
FluorescenceDetection
Kias Undgren1 and Nikos Rodopoul&
We describe
a chromatographicprocedure for sensitive (2.0
1znlol/L), specific quantification
of vanillylmandelic acid
(VMA) in urine with iso-VMA as an internal standard.After
rapid extraction from urine, the VMA is determined by
isocratic
reversed-phase ion-pair chromatography on a
bonded C8 column and detectionof the native fluorescence
on excitation at 285 nm. The fluorescence signal is quite
dependent on the pH of the mobile phase. Results by the
method vary linearly with VMA concentration upto 320 jzmov
L and correlate well (r = 0.9880) with those obtained by
conventionalultravioletspectrophotometry.The mean 24-h
excretionof VMA from 29 healthy volunteers was 21.4 (SD
5.4) zmol.
AddItIonal
chromatography, reversed-phase
urine
reference values
screening
Keyphras.s:
variation,source of
#{149}
Department of Clinical Chemistry, Danderyd’s Hospital, 8-18288
Danderyd, Sweden.
Present addresses: ‘Institute of Medical Microbiology, Department of Clinical Bacteriology, Guidhedagatan 10,8.41346 Gothenburg, Sweden. 2Department of Clinical Chemistry, Beckomberga
Hospital, S-161 04 Bromma, Sweden.
Received December 4, 1985; accepted January 22, 1986.
One of the principal
metabolites of epinephrine
and
norepinephrune that is excreted in urine is vanillylmandelic
acid (VMA), and its quantification
is of important aid in the
diagnosis of pheochromocytoma
(1, 2). VMA has been
determined
colorinietrically
(3), by paper chromatography
(4), and by thin-layer chromatography
(5,6). However, these
methods are time consuming,
lack specificity, and are
difficult to perform quantitatively.
Gas chromatographic
methods (7-9) are specific and sensitive, but require derivatization, which also is too time consuming and complicated
for routine use. “High-performance”
liquid-chromatographic
(HPLC) procedures have been used, with ultraviolet spectrephotometric
(10, 11), electrochemical
(12-Jo), and fluorescence detection (16).
Among the various compounds used as internal standards
in chromatographic
assays of VMA (8,9,11), 3-hydroxy-4methoxymandelic
acid (iso-VMA) has been widely used in
HPLC. Here we describe an HPLC procedure with fluores..
cence detection, which offers the advantage
of chromatographic stability and selectivity. Iso-YMA is used as the
internal standard, for high precision and accuracy. Because
3Nonstandard abbreviations: VMA, vanillylmandelic acid, isoVMA, 3-hydroxy-4..methoxymandelic acid; HPLC, “high-performance” liquid chromatography; TBA, tetrabutylammonium
hydrogen sulfate.
CUNICAJ..
CHEMISTRY,Vol.32, No.4, 1986 693