Effect of TrIton X-100 on
PrecipItation of Biliary Phosphatase
by Wheat-Germ Lectin
To the Editor:
The recent paper by Sorensen (1)
prompts us to draw attention to our
previous Letter (2),which pointed out
that, to prevent precipitation
of biliary
phosphatase in our wheat-germ
lectin
precipitation
method for alkaline phosphatase (ALP, EC 3.1.3.1) (3), we preincubated plasma samples with an
aqueous solution of Triton X-100 surfactant of concentration 200 g/L, not 20
g/L, added in the proportion 5 L of
Triton X-100 to 50 pL of plasma to
yield a pre-incubation concentration of
Triton X-100 of 18 g/L. The lower Triton X-100 concentration is inadequate
to fully prevent biliary ALP precipitation (Figure 1).
As further
indicated
in our Letter,
the inconvenience of the pre-incubation step may be avoided by including
Triton X-100 in the precipitation reagent. We use a concentration of 40 g/L
so that, when it is mixed with an equal
volume of plasma, the final concentration of Triton X-100 in the incubation
mixture is 20 g/L.
2. Rosalki SB, Foo AY. Simplified wheat
germ lectin precipitation method for alkaline phosphatase isoenzyme. [Letter]. Chin
Chem 1986;32:2118.
3. Rosalki SB, Foo AY. Two new methods
for separating and quantifying bone and
liver alkaline phosphatase isoenzymes in
plasma. Clin Chem 1984;30:1182-6.
Sidney B. Rosalki
A. Ying Fee
Dept. of Chem. Pathol. and Human
Metab.
The Royal Free Hospital and School of
Medicine
London NW3 2QG, UK.
B I
2
* WG tIn
3
Un5..d
*
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&
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,.fl,,
References
1. Yates P, WeinkoveC. Simplificationof a
urinary metadrenahine assay. Ann ChinBiochem 1986;23:487-9.
2. Vanley H, Gowenlock AH, Bell M. Practical clinical biochemistry, Vol. 2, 5th ed.
London: Heinemann,
1976:206.
Lynn Rowbotham
Angus Molyneux
Brian Morris*
Modified Method of Analysis for
Metadrenallne In Urine
To the Editor:
We describe here some modifications
to a previously reported method for
determination of metadrenaline in human urine. The method of Yates and
Weinkove (1) is a useful addition to the
routine laboratory repertoire for investigating pheochromocytoma.
However,
in our experience,
the measured absorReferences
bances are frequently very low, which
1. Sorensen S. Wheat-germ agglutinin
can adversely affect precision. The
method for measuring boneand liver isoenmain prerequisite for reproducible rezymes of alkaline phosphataseassessedin
suIts is the addition, in slurry form, of
postmenopausal osteoporosis. Clin Chem
a constant
quantity
(5 mL) of CG 50
1988;34:1636-40.
resin (Sigma Chemical Co., Poole, Dorset, U.K.). This is difficult to achieve in
practice, and resin volumes may vary
#{149}
WcI.cun
from 3 to 5 mL between samples, with
consequent variable diminution of the
final absorbance.
Pr5b
I
We have attempted to improve the
sun
precision of resin volume by drying the
5 #{149}WGI.,**21xIT
resin after activation (2)and adding it
in powder form (2.5 g per tube, equiva7 #{149}WCI.tin
lent to 5 mL of slurry). This reduces
considerably the between-tube variation in resin content, and thereby proFig. 1. ALP electrophoretic separation patternsobtained before and after precipitation vides a constant number of binding
sites in each tube. Further, we have
with wheat-gem, lectin
Tracks 3 and 4 show anuntreatedserumcontaining increased the reaction times for both
liver and biltaty (ol) ALP. Precipitationwascarried the initial resin binding and final amout with an equal volume of an aqueous solution of
momacal wash from 20 to 30 mm, and
wheat.9ermlecth, (5 g/L), withor withoutTritonXuse three wash cycles rather than one.
100addedto yielda finalconcentrationof20 g/Lor2
g/L. The compositionof the supemates is shown in In our hands, this yields final absorthe upper half of the figure.Even with the higher bances two or more times greater than
Triton X-100 concentration(track 1), billary ALP is those attainable
by the published
bound,but not precipitated,andis visibleas a band method.
at the origin.UsingN-acetylglucosamine(10 g/L)to
Analytical recovery of metadrenaelute ALP from the precipitates(lower half of the
line in this modified method was befigure) revealsthe presenceof biliaryALP in the
precipitateresulting from addition of wheatlerm
tween 80% and 102% for concentralectinalone (track ?)or with the lowerconcentration tions between 1 and 20 tmoIJL. Withof Triton X-100 (track , but not with the higher
TritonX-100 concentration(track 5); i.e.,the higher in-batch CVs were 9% at 5.3 mol/L
and 4% at 13 .imol/L (n = 10). Beconcentration
of Triton X-100 (finalconcentration
20
g/L)is neededtopreventsuch precipitation
tween-batch CV was 14% at 2.8 anol/
* 2.1I5
L (n = 8). A 95% reference interval of
1.5-7.5 .tmo1J24 h was established by
assaying 24-h urine collections from 12
healthy volunteers.
We recommend the use of these
three simple modifications, which in
our experience noticeably improve the
magnitude
and precision of the final
absorbance.
Clin. Chem. Dept.
Northern General Hosp.
Herries Rd.
Sheffield, S5 7AU
S. Yorkshire, U.K.
*Cormspondingauthor.
Incorporation of Butylated
Hydroxytoluene in the Mobile Phase
increases Sensitivity in Carotenoid
Chromatography
To the Editor:
Thurnhazn
et al. (1) reported that
the sensitivity of their assay for plasma carotenoids was occasionally adversely affected by the methanol in the
mobile phase. We recently set up this
method in Chiangmai, Thailand, but,
with available reagents, found it possible to measure carotenoids only at concentrations 10-fold those found in human blood. We were using “liquid chromatography”-grade
methanol from
Merck (F.R.G.), but alternative supplies from J.T. Baker Co. (U.S.A.) or
glass-redistilled methanol also failed to
improve sensitivity. However, the
incorporation of a small amount of
butylated hydroxytoluene (BHT) in the
mobile phase had an enormous effect
on analytical recovery of carotenoids.
The equipment used and the basic
methodology are described elsewhere
(1).
The mobile phase (acetomtrile/
methanolichloroform,
47/47/6 by vol)
was pumped at 0.5 mL/min through a
125 x 4.6 mm column of 3-jnn particles of Spherisorb ODS-2 (PharmaciaUK Ltd). Retinol
(325 mn) and a-tocopheroh (292 nm) in the eluate were
CLINICAL CHEMISTRY, Vol. 35, No. 3, 1989
513
without any problem, but no
absorbance at 450 nm was detectable
whether carotenoid standards
or serum extracts were used. After we incorporated 10 mg of BHT per liter of
mobile phase, however, subsequent
fractionations
of carotene standards
produced increasing amounts of absorbance at 450 nm at the expected retention times. Once the elution pattern
had stabilized, we attempted to determine the optimal concentration
of BHT
for carotenoid chromatography
but
found that concentrations between 5
and 100 mg/L did not change the chromatography. Nor did omitting BHT
from the mobile phase completely for
short periods (30 mm) interfere with
subsequent carotenoid
chromatogrameasured
phy.
Our experience is with 3-sm columns where we and others have incorporated BHT into the extraction proce-
dure (1,2). Other workers using 5-.an
columns for carotenoid
chromatography do not report using BHT (3-5).
Our observation concerning BHT may
only be applicable to 3-zm columns;
however, they may help to explain
some of the instability experienced by
various workers in carotenoid chromatography.
Small amounts of BHT in
sample extracts may suffice to exert
the stabilizing effects on the column;
hence these observations have not
been reported before. In our previous
experience some supplies of methanol
and the use of the cartridge
guard
column caused poor sensitivity
with 3tm Pharmacia columns (1), while 3zm Hypersil columns showed declining sensitivity with increasing retention times (unpublished results). These
new observations suggest that BHT is
interacting
with some component
within the column itself to protect carotenoids, and this interaction would
not appear to be easily reversible when
the column is eluted without BHT.
D.I.T. is grateful for continuing support
from the Dept. of Health & Social Security,
U.K.
References
1. Thurnhani DI, Smith E, Flora PS. Concurrent liquid-chromatographic assay of
retinol,
a-tocopherol,
/3-carotene, a-caro-
tene, lycopene, and /3.cryptoxanthin in plasma with tocopherol acetate as internal standard. Clin Chem 1988;34:377-81.
2. Milne DB, Botnen J. Retinol, a-tocopherol, and a- and p-carotene simultaneously
determined in plasma by isocratic liquid
chromatography. Clin Chem 1986;32:8746.
3. Vuilleumnier J-P, Keller HE, Gysel D,
Hunziker F. Clinical chemical methods for
the routine assessment of the vitamin status in human populations. Part I: The fat514
soluble vitamins A and E, and /3-carotene.
bit J Vitam Nutr Res 1983;53:265-72.
4. Broich CR, Gerber LE, Erdman Jr JW.
Determination of lycopene, a- and p-carotene and retinyl esters in human serum by
reversed-phase high performance liquid
chromatography. Lipids 1983;18:253-8.
5. Craft NE, Brown ED, Smith Jr JC. Effects of storage and handling conditionson
concentrations of individual carotenoids,
retinol, and tocopherol in plasma. Clin
Chem 1988;34:44-8.
R. Singkamani
S. Koottathep
A. Supapang
Res. Instit. for Health Sciences
Chiang Mai University
P.O. Box 80 CMU, Chiang Mai 50002
Thailand
D. I. Thurnham
Med. Res. Council, Dunn Nutrition
Laboratoiy
Downhams Lane, Milton Road
Cambridge CB4 JXJ
England
Salivary Melatonin Estimation in
Assessment of Pineal-Gland
Function
To the Editor:
The pineal-gland hormone, melatonm, is now considered a clinically useful component
of neuroendocrine
physiology (1). The relevance of investigation of human pineal-gland function via melatonin assay has been emphasized by the findings of disturbances in melatomn secretion in a
variety of disease states (2,3). This
hormone is associated, both diagnostically and etiologically, with sex-steroid-responsive neoplasia of the breast
and prostate (4, 5), and there may be
similar diagnostic potential and etiological significance for major affective
disorders (6). Research into the physiological significance
and clinical utility
of melatonin in humans has been limited by the practical (and sometimes
ethical) problems associated with the
study of the concentrations of this nocturnally
secreted hormone in plasma.
To facilitate long-term chronobiological-type studies of the basic and clinical importance of melatonin,
we developed a direct RIA for salivary melatonm (7)based on some principles of an
existing plasma assay (8). Subsequent
studies, all published in this journal
(for review, see 3 and 9) indicated a
highly significant, reproducible corre-
CLINICAL CHEMISTRY, Vol. 35, No. 3, 1989
lation between plasma and salivary
concentrations of melatonin in simultaneously collected samples taken during a 24-h period. Subsequent confirmatory studies by independent investigators yielded results that agreed well
with our original observations (10,11).
A matter of fundamental
concern
regarding the utility of salivary melatonin assay in basic and clinical investigations of human pineal-gland function, has been the effect of variations in
salivary flow rate on salivary melatonm concentrations. McIntyre et al. (10)
could demonstrate no disruption of the
plasma-saliva melatonin ratio when
saliva was collected during maximal
stimulation of flow rate. Similarly, Nowak et al. (11) found no disruption of
this ratio when they collected saliva
without stimulating the flow rate. In
one of our preliminary studies (12), we
detected a highly significant effect of
variations in flow rate on salivary melatonin concentration,
but later found
this effect on the RIA system to be due
to potato-chip contamination
of saliva.
The effect of contaminants
of saliva on
salivary melatonin assay has been noted by others (10), and it emphasizes the
need to adhere to strict guidelines in
the collection of saliva samples. Further highly standardized experiments
by our group demonstrated no significant effect of salivary flow rate on
salivary melatonin concentrations.
We
report here the results of a study aimed
at investigating the effects of flow rate
on salivary melatonin concentrations.
Ten subjects, five men (19-33 y) and
five women (20-25 y), participated
in
the study after ethical approval and
informed consent had been obtained.
Blood was removed by venesection at
0100 hours and 0700 hours, and the
plasma was separated by immediate
centrifugation, thus precluding the necessity for anticoagulants. Saliva (2.5
mL) was collected without stimulation,
as described previously (7),immediately before venesection, and saliva was
collected during maximal stimulation
of flow rate immediately after venesection. The volunteers maximally stimulated their salivary flow rate by chewing sterilized rubber bands. Salivary
melatonin was estimated by direct RIA
(7), plasma melatonin by the method of
Fraser et al. (8).
The results (Table 1) show no significant difference in the concentration of
melatonin in stimulated vs unstimulated salivas, during times of peak
melatonin secretion and times it was
declining from nocturnal
values, nor
were significant
alterations
in the
plasma-saliva
melatonin ratio observed.