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Biotinidase determination in serum and dried blood spots - High

sensitivity fluorimetric ultramicro-assay

Article  in  Clinica Chimica Acta · January 2002

DOI: 10.1016/S0009-8981(01)00690-8 · Source: PubMed

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 Clinica Chimica Acta 314 Ž2001. 175–185
www.elsevier.comrlocaterclinchim

Biotinidase determination in serum and dried blood spots—high

sensitivity fluorimetric ultramicro-assay
Elke Broda a , E. Regula Baumgartner b, Sabine Scholl c , Marina Stopsack d ,
Anton Horn a , Heidrun Rhode a,)
a
Institute of Biochemistry, Medical Faculty, Friedrich Schiller UniÕersity, D-07740 Jena, Germany
b
Metabolic Unit, UniÕersity Children’s Hospital, CH-4005 Basel, Switzerland
c
Medical School HanoÕer, Children’s Hospital, D-30625 HanoÕer, Germany
d
New-born Screening Centre, Medical Faculty, Carl GustaÕ Carus UniÕersity, D-01307 Dresden, Germany
Received 13 March 2001; received in revised form 9 August 2001; accepted 16 August 2001

Abstract

A miniaturized quantitative biotinidase assay has been developed using biotin 6-amidoquinoline as substrate and the
100-fold enhanced fluorescence of 6-amidoquinoline measured using apolar solvents. Amidoquinoline is measured after
deproteinization by ethanolracetone using individual standardisation and solvent resistant microtiter plates. The assay was
optimized for end point determinations of biotinidase activities in serum and for newborn screening using dried blood spots.
Serum activities obtained are closely correlated with values obtained using a quantitative validation method Ž r s 0.96..
Analytical sensitivity is around 2% of the mean activity Ž7.01 " 1.92 nmolrminrml, mean " SD. of a healthy control
population.
With dried blood spots, a close correlation with values obtained using the Wallac-test kit Ž r s 0.92. was found.
Biotinidase activities of a healthy population of 651 newborns amount to 0.2429 " 0.07 nmolrminrml blood. The analytical
sensitivity is close to 1% of the mean activity. q 2001 Elsevier Science B.V. All rights reserved.

Keywords: Biotinidase deficiency; Screening; Fluorimetry

1. Introduction product of the four reported biotin-dependent car-

Biotinidase ŽEC 3.5.1.12. recycles the vitamin
biotin by liberating it from biocytin, the degradation
AbbreÕiations: AQ, 6-amidoquinoline; BAQ, biotin 6-ami-
doquinoline; MES, Morpholinoethansulfonic acid; Tris, Tris
Žhydroxymethyl.aminomethan; MOPS, 3-Ž N-morpholino.pro-
X
pansulfonic acid; HEPES, N-2-hydroxyethylpiperazin-N -2-ethan-
X Y
sulfonic acid; BisrTris, 2,2-Bis-Žhydroxymethyl.-2,2 ,2 -nitrilotri-
ethanol.; FU, relative fluorescence units.
)
Corresponding author. Tel.: q49-3641-938634; fax: q49-
3641-938612.
E-mail address: hrho@mti-n.uni-jena.de ŽH. Rhode..
boxylases. Biotinidase has been shown to have bi-
otinyl-transferase and transporting activities, too w1x.
Biotinidase deficiency is a rare, autosomal reces-
sively inherited disorder of biotin metabolism result-
ing in an inability to recycle biotin from endogenous
sources or to utilize biotin bound to dietary proteins.
Clinical symptoms as well as time of onset vary
greatly, resulting in deficiencies of the biotin depen-
dent carboxylases to various degrees w2–6x. Bio-
tinidase deficiency meets all WHO screening criteria
and is one of the inborn errors of metabolism recom-
mended for inclusion in extended neonatal screening

0009-8981r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 0 0 9 - 8 9 8 1 Ž 0 1 . 0 0 6 9 0 - 8
176 E. Broda et al.r Clinica Chimica Acta 314 (2001) 175–185
w7x. Due to the yet incomplete screening in a variety
of countries, only rough estimates of incidence data
are available ranging from 1:33 000 w8x to 1:500 000
w9x for profound biotinidase deficiency Ž0–10% of
the mean normal activity., and ranging from 1:14 000
w8x to 1:129 000 w10x for partial deficiency Ž10–30%
activity..
If other metabolic processes or poor nutrition lead
to increased requirements for biotin, a partial defi-
ciency also may cause chronic infections w11x as well
as dermatological and neurological symptoms w12x.
Biotinidase determination was facilitated by the
colorimetric method of Knappe et al. w13x using
biotinyl p-aminobenzoate as substrate. This method
was adapted also to screening purposes w14,15x. The
enzyme activity may be determined by radiochemi-
cal assays w16,17x, by time-resolved fluorimetry w18x,
and by a microbiological bioassay w19x. The fluori-
metric assay using biotin 6-amidoquinoline ŽBAQ.
was introduced by Wastell et al. w20x. BAQ has been
used for screening serum activities w21–24x and, in
combination with deproteinization, for newborn
screening with dried blood spots in microtiter plates
w25x.
Surprisingly, we found that the fluorescence of
6-amidoquinoline measured at 440 nm and in the
presence of apolar solvents was stronger by two
orders of magnitude than that measured at 540 nm,
the commonly used wavelength. For the present
work, Wastell’s method was improved using these
new fluorescence measurement conditions. Adapted
multiwell plates are used; an improved deproteiniza-
tion and individual fluorescence standards were in-
troduced. This results in higher sensitivity at lower
costs by reduction of the final assay volumes to
15–20 ml. The method can be used with slight
modifications with serum and dried blood spots.
2. Materials and methods
2.1. Chemicals
Dithiothreitol was from Serva. Biotin 6-amido-
quinoline was purchased from Sigma. 6-Amidoqui-
noline was from Aldrich. The Wallac test ANeona-
tal Biotinidase Test kitB ŽNB-1000E. was from
PerkinElmer Wallac Inc. MN 818 filter paper sheets
and Guthrie cards were obtained from Macherey and
Nagel. All other reagents were of analytical grade
and purchased from Fluka, Sigma, or Merck.
2.2. Samples
Control serum: Serum from 936 apparently healthy
blood-donor volunteers, from the regional Transfu-
sion Service Žfemalermale: 440:502; mean age
Žyears " SD.: 36.7 " 13.2; range: 18–67 years..
Control blood spots: unused dried blood spots from
Guthrie cards of 651 normal newborns from the
screening centre of the Carl Gustav Carus Univer-
sity, Dresden. The samples were checked before use
by the semiquantitative method according to Ref.
w14x and were used in our laboratory within 14 days
after collection.
Biotinidase deficient samples: Spare blood and
serum samples from biotinidase deficient children
were supplied by the Metabolic Unit of the Children’s
Hospital of the University Basel and the Children’s
Hospital of the Medical School Hanover. The sam-
ples were checked before use according to Ref. w26x.
Dried blood spots were obtained from the Children’s
Hospital Leipzig or were prepared from whole blood
samples.
2.3. Experimental design
Five milligrams of BAQ were dissolved in 0.5 ml
ethanol. This solution can be stored at 4 8C or at
y20 8C for several weeks. Pipetting was done using
8- to 12-multichannel pipettes or the 96-channel
pipette ACybi-wellB ŽCybio Jena.. Fluorescence was
read in multiwell plates using the Fluoroskan II
ŽLabsystems.. Quantitative absorbance and fluores-
cence measurements were made using a Beckman
DU w-70 and a Perkin Elmer LS 50B device, res-
pectively. After deproteinization, centrifugation of
multiwell plates was performed in an Eppendorf
Centrifuge 5403 at 4000 rpm for 5 min. Ultramicro-
multiwell screening plates were obtained from Vaku-
form Scharff, D-99887 Catterfeld w27,28x.
For comparison, biotinidase activities of some
samples of whole blood and dried blood spots were
determined according to Yamaguchi et al. w29x.
 E. Broda et al.r Clinica Chimica Acta 314 (2001) 175–185 177

3. Results fluorescence increased proportionally to AQ concen-

3.1. Fluorescence spectra of 6-amidoquinoline (AQ)
and biotin 6-amidoquinoline (BAQ)
Intensity and spectrum of AQ fluorescence de-
pend strongly on the presence and the concentration
of organic solvents ŽFig. 1.. From the solvents tested,
ethanol, ethanolracetone, and methanolracetone
Žboth mixtures 1:1, vrv. enhance the intensity of the
first fluorescence maximum observed in the range of
438–450 nm by about two orders of magnitude
ŽTable 1.. The second, commonly used, fluorescence
value of around 530–540 nm also rises slightly with
increasing solvent concentration but the maximum
flattens out and disappears at higher solvent concen-
tration. The excitation maximum of around 350 nm
did not shift in the presence of solvents but also
increased with solvent concentration. In the presence
of 80% ethanolracetone, the fluorescence intensity
measured at 450 nm increased with pH in the range
from 5.0 to 6.5 and showed a maximum between pH
6.5 and 9.0 Ždata not shown.. In multiwell plates, the
tration up to about 100 mmolrl. The fluorescence of
BAQ is very weak and was not altered significantly
by any solvent at 355-nm excitation and 440–460-nm
emission Žnot shown.. Therefore, the biotinidase re-
action product AQ should be measured around 440
nm Ž355-nm excitation..
3.2. Deproteinization
Since determination of AQ is disturbed by the
strong fluorescence quenching effects of serum pro-
teins, and especially by hemoglobin, these proteins
have to be removed from the reaction mixture. Both
the removal of proteins and the enhancement of the
fluorescence signals may be achieved by the same
agent. The deproteinization effects of organic solvent
mixtures were determined by absorbance measure-
ments at the wavelengths used for fluorimetric AQ
determination and at the concentrations used with
assay mixtures in multiwell plates, such as 75–85%
organic solvent as shown in Table 2. With serum and
with blood spots, methanolracetone and ethanolr

Fig. 1. Fluorescence spectra of 6-amidoquinoline ŽAQ. in aqueous media and in media containing organic solvent. Fluorescence of AQ was
measured with excitation at 355 nm in 150 mmolrl MESrNaOH, pH 6.5 using a Perkin Elmer LS 50B device. The content of the organic
solvent ethanolracetone Ž1:1, vrv. was varied. v, without organic solvent, shown in the inset; l, with 30% Žvrv. organic solvent; B,
with 60% Žvrv. organic solvent, and ', with 80% Žvrv. organic solvent. Notice the different concentrations of AQ used, namely 9.71 and
0.971 mmolrl for 0–30% solvent and for 60–80% solvent, respectively.
178 E. Broda et al.r Clinica Chimica Acta 314 (2001) 175–185

Table 1
Maximum fluorescence intensities, fluorescence ratios, and peak wavelengths of AQ in aqueous solutions and in mixtures containing
organic solvents
Solvent addedr Maximum fluorescence l Žnm. of the Ratio of fluorescence Ratio of fluorescence
concentration Ž%vrv. intensities ŽFUrmmolrl. a emission maximum values Ž f 1rf 2. b values Ž f 1rf535.5 . c
Without solvent 3.6 535.5 0.45 1.0

Ethanol
30 10.8 448.5 1.64 3.0
60 77.5 442.5 7.70 21.3
95 375.3 440.5 24.80 103.4

Ethanolr acetone
30 11.9 446.5 3.08 3.3
60 189.9 445.0 16.04 52.8
80 444.4 443.0 27.03 123.4

Methanolr acetone
30 11.5 447.5 1.94 3.2
60 88.4 444.0 7.27 24.4
95 315.8 438.0 20.53 87.0

Fluorescence measurements of AQ were performed in 150 mmolrl MESrNaOH, pH 6.5 containing 1.18 mmolrl dithiothreitol using a
Perkin Elmer LS 50B at 355-nm excitation. Different AQ concentrations were used to get intensities that could be reliably measured.
a
Fluorescence intensities measured in the medium indicated in column 1 at the wavelengths indicated in column 3. The fluorescence
values given are normalized by the AQ concentration.
b
Ratio of the fluorescence intensities measured in the medium indicated in column 1 at the wavelengths indicated in column 3 Ž f 1. and
of the fluorescence intensities measured at 538 nm in the same medium Ž f 2..
c
Ratio of fluorescence intensities measured in the medium indicated in column 1 at the wavelengths indicated in column 3 Ž f 1. and of
the fluorescence intensity measured without solvent at 535.5 nm Ž3.6 FU..

Table 2
Deproteinization of blood plasma and blood spot eluates by organic solvents
Protein source and Deproteinization Final concentrations Absorbance Absorbance Absorbance Absorbance
buffer A concentration solvent Žsolvent Žbuffer Arsolvent 1r at 355 nm at 460 nm at 355 nm Ž%. at 460 nm Ž%.
Žmmolrl. 1rsolvent 2. solvent 2, vrvrv.
Plasma
150 none 1r0r0 0.134 0.109 100 100
ethanol 1r4r0 0.046 0.051 34.0 47.2
ethanolrr acetone 1r2r2 0.019 0.051 14.0 46.9
methanolracetone 1r2r2 0.024 0.037 18.0 33.9

Dried blood spots

150r100r75 none 1r0r0 5.418 " 0.12 1.793 " 0.19 100 100
150 ethanol 1r3r0 0.841 0.351 15.9 22.0
methanolracetone 1r1.5r1.5 0.392 0.242 7.4 15.2
ethanolracetone 1r1.5r1.5 0.347 0.140 6.6 8.8
100 ethanolracetone 1r1.5r1.5 0.220 0.086 4.0 4.3
75 ethanolrr acetone 1r1.5r1.5 0.200 0.078 3.6 4.3

One hundred sixty microliters of plasma was diluted with 600 ml of buffer A Ž150 mmolrl MESrNaOH, pH 6.5 containing 1.5 mmolrl
dithiotreitol.; 3.0 ml of either buffer A or of the solvents indicated in column 1 were added. One hundred sixty blood spots ŽB 3 mm. were
eluted with 3.2 ml of buffer A overnight at room temperature. Thereafter, 2.1 ml of either buffer A or of the solvent were added to 0.7 ml
aliquots of the eluate. The mixtures were vortexed and centrifuged at 4000 rpm for 5 min. The absorbance of the supernatants was read
using appropriate dilutions and compared with those of blanks prepared in parallel without sample. Bold entries indicate deproteinization
conditions used for standard assays.
 E. Broda et al.r Clinica Chimica Acta 314 (2001) 175–185 179

Table 3 ment Žsee Table 1.. As the removal of quenching

Standard assay conditions for biotinidase determination with serum compounds is still incomplete, individual fluores-
and dried blood spots
cence standards have been introduced for biotinidase
Assay constituents and Serum Dried blood
measurements Žsee Section 3.4..
conditions spot
Sample size 4 ml B 3 mma
Volume of test mixture 15 ml 20 ml
added
3.3. Optimization of the biotinidase assay
Buffer A concentration 150 mmolrl 75 mmolrl
BAQ concentration 675 mmolrl 675 mmolrl
Incubation time 2h 17 h
The enzymatic assay conditions were optimized
Incubation temperature 38 8C 18–24 8C differently to meet the requirements of miniaturized
Volume of ethanolracetone 75 ml 60 ml tests for serum and blood spot samples. Various
Ž1:1, vrv. added buffer substances, e.g. Tris, MOPS, MES, HEPES,
a BisrTris were tested at 150 mmolrl and at pH 6.5.
One dried blood spot of 3-mm diameter corresponds to about
4 ml of whole blood as calculated from diameters produced by 50 From all buffers tested, MES produces the strongest
ml of blood. biotinidase signals with both kinds of samples. The
optimum pH for both kinds of samples is 6.5. The
deproteinization conditions finally used are indicated
in Table 2.
acetone produce the strongest deproteinization, re- The K m of serum biotinidase with BAQ as sub-
spectively. Nevertheless, ethanolracetone was cho- strate was found to be 28.3 mmolrl as determined
sen for measurements with both samples because it under standard assay conditions as shown in Table 3.
produces the strongest AQ-fluorescence enhance- The fluorescence increase is proportional to bio-

Table 4
Imprecision of the new biotinidase assay with various types of samples
Type of sample Donor Relative Mean activities and imprecisions
number activity Ž%. a Intra-assay Inter-assay
Mean activity CV n Mean activity CV n
Žnmolrminrml. Žnmolrminrml.
Serum 1 1.2 0.084 13.27 32 – – –
2 1.6 – – – 0.11 34.8 8
3 4.2 – – – 0.29 14.2 8
4 26.5 1.86 4.73 32 2.22 14.3 21
5 45.8 3.21 5.18 16 3.27 18.2 7
6 119.7 8.39 2.83 32 – – –
7 140.9 – – – 9.87 11.5 13
Dried blood spot 2 1.8 0.01 16.8 24 – – –
4 26.3 0.06 7.88 32 0.06 15.0 21
8 34.6 – – – 0.08 12.5 16
9 131.7 0.32 6.70 28 – – –
7 161.0 – – – 0.39 16.9 16
10 190.6 – – – 0.46 15.0 20

The intra-assay imprecision was determined with single determinations on n positions of one multiwell plate. The inter-assay
imprecision was obtained from single measurements on n days. Because only small sample volumes were available from some biotinidase
deficient patients, different samples were used from such patients to cover the whole activity range. Note the very low activities of dried
blood spot biotinidase. The activities given were determined after drying and transport in comparison to AQ standards Žcf. Section 3.8..
a
Biotinidase activity of the donor related to the mean activity of the corresponding cohort, i.e. blood donor sera and newborn dried blood
spots.
180 E. Broda et al.r Clinica Chimica Acta 314 (2001) 175–185

Fig. 2. Serum biotinidase activity determination: Correlation between results of the standard assay and the assay according to Ref. w26x.
Thirty prevalidated serum samples selected such as to cover the whole activity range were tested in parallel with both assays. Profound Ž`.,
partial ŽB. and no biotinidase deficiency Žv . are marked.

tinidase concentration and linear with time for 2 and
6 h at 37 8C with normal serum and dried blood
spots, respectively. Normal serum biotinidase activ-
ity exhibits an RQ10 value of 2.22 as calculated from
incubations between 23 and 39 8C Ždata not shown..
3.4. Standard assay conditions
The standard assays with punched blood discs or
serum are each performed as an incubation set con-
sisting of one biotinidase test, one individual blank,

Fig. 3. Biotinidase activities determined from dried blood spots: Correlation between results obtained with the standard assay and the Wallac
test kit. Forty-one dried blood spots selected such as to cover the whole activity range were tested in parallel with the standard assay and
with the test kit supplied by Wallac. The different ranges of activity values obtained with both tests can be explained by the different
calibrators used. Our results were obtained by comparison with the AQ-standard whereas the commercial test kit uses calibrator spots of
known original blood biotinidase activity.
 E. Broda et al.r Clinica Chimica Acta 314 (2001) 175–185 181

and one individual standard on the same multiwell ferent newborn dried blood samples showing differ-
plate. Three samples of serum or dried blood spots ent biotinidase activities. Analytical sensitivity is
from each proband were placed each on the bottom defined as the sum of the mean fluorescence of all
of one well of the plate. Biotinidase test incubations individual blanks plus 3 SD.
are performed with buffer A, BAQ, and 1.5 mmolrl No false positive or false negative results were
dithiotreitol Žsee Table 3.. Individual blanks are ob- found with the small number of biotinidase deficient
tained using the same conditions but with ethanol samples at hand and with dried blood spots of 651
instead of the ethanolic BAQ stock solution. For newborns.
individual standards, 97.1 mmolrl AQ in buffer A is Biotinidase activity values from serum and dried
added to a third sample. The incubations were started blood spots were compared to values obtained using
by simultaneous addition of the test mixtures, blank the assay according to Ref. w26x and the semi-quanti-
mixtures and standard mixtures. The plates were tative Wallac test kit w25x, respectively. Results of
covered with plate sealers and gently shaken using a correlation analyses are shown in Figs. 2 and 3. The
microtiter plate shaker for 1 min. Biotinidase reac- measured fluorescence values obtained after 17 h
tions with serum and blood spot samples were per-
formed at 38 8C and at ambient temperature, respec-
tively, for the times given in Table 3.
Biotinidase activities of sera and dried blood spots
are evaluated from end point measurements at 460
nm Ž355-nm excitation. after termination by depro-
teinization using cold ethanolracetone Ž1:1, vrv..
After shaking for 1 min, the plates were centrifuged
using a microtiter plate rotor. Biotinidase test incuba-
tion, blank incubation, and AQ standard incubation
produce test fluorescence T, blank fluorescence b,
and standard fluorescence S, respectively. The bio-
tinidase activities B were calculated using the fol-
lowing formula:
B s D 97.1 nmolrml= Ž T y b . r Ž S y b . rt
where: t, time in minutes; D, dilution factor Ž19r15
with serum samples, 20r4 with dried blood spot
samples..

3.5. Imprecision

Table 4 shows imprecisions found with samples

of different types.

3.6. SensitiÕity, comparison with other tests

The analytical sensitivity of biotinidase measure-

ment with serum and dried blood spots is 0.165
nmolrminrml Ž2.35% of the mean activity in serum
of blood donors. and 0.0019 nmolrminrml Ž0.8%
of the mean activity in dried blood newborn sam-
ples., respectively. This parameter was determined
from measurements of 30 different sera and 26 dif-
Fig. 4. Frequency distribution of biotinidase activities. ŽA. Serum
biotinidase activities of 936 adult healthy blood volunteers. Bio-
tinidase activities of 936 serum samples of the regional blood
transfusion service were determined using standard assay condi-
tions. ŽB. Dried blood spot activities of 651 healthy newborns.
Samples were stored after use by the screening centre and trans-
ferred to our laboratory in dry ice.
182 E. Broda et al.r Clinica Chimica Acta 314 (2001) 175–185

incubation at 38 8C using the Wallac test amount to
one fiftieth of those obtained by the standard assay
presented here.
3.7. Biotinidase actiÕities of Õarious populations
3.7.1. Normal serum actiÕities
The control population of 936 randomly selected
healthy blood donors exhibits a broad biotinidase
activity range from 2.15 to 16.04 nmolrminrml
serum, mean " SD being 7.007 " 1.92 nmolrminr
ml ŽFig. 4A.. One specimen with partial biotinidase
deficiency Ž2.15 nmolrminrml, - 30% of the mean.
was found representing 0.107% of the cohort tested.
3.7.2. Dried blood spots from newborns
Biotinidase activities of 651 dried blood spots
from healthy newborns were tested about 1 week
after routine testing by the screening centre. The
activities obtained also cover a broad range of 0.07–
0.64 nmolrminrml blood with a mean of 0.243 "
0.07 nmolrminrml as shown in Fig. 4B. No sample
exhibiting biotinidase activity below 30% of the
daily mean activity was found.
3.8. Sample stability
Stability of biotinidase activity was tested in both
specimens under study and at different storage tem-

Fig. 5. Stability of biotinidase activity. Biotinidase activity found with serum: Aliquots of serum of a healthy volunteer were tested in
duplicates immediately after withdrawing and after storage for various periods at different temperatures. Biotinidase activity found with
dried blood spots: Biotinidase activity of dried spots ŽB 3 mm. of a healthy volunteer was tested using standard assay conditions after
storage at different temperatures and for different periods. Blood spots were prepared by dropping 50 ml of whole blood onto filter paper
sheets ŽMN 818.. All dried samples were stored in tight Zip-bags.
 E. Broda et al.r Clinica Chimica Acta 314 (2001) 175–185
peratures. The enzyme activity is stable in serum and
in dried blood spots at y20 and y80 8C for more
than 2 months and for at least 5 months, respec-
tively. Storage of all samples at room temperature
and at 4 8C produces a significant activity loss with
time as shown in Fig. 5.
4. Discussion
4.1. AQ measurement
At present, AQ is measured at 538 nm in a variety
of tests irrespective of the solvent composition.
However, we found the fluorescence properties of
AQ strongly dependent on the presence of organic
solvents. Tests using AQ as an analyte yielded much
higher sensitivities when measured at 440–460 nm
w30x. Therefore, the reaction product of biotinidase
may be detected much more sensitively at 440 nm
Ž355-nm excitation. than with the commonly used
wavelength combination Ž355r538 nm.. With the
Fluoroskan II, the filter combination 355 " 35r460
" 25 nm fulfils these demands sufficiently.
Biotinidase produced AQ fluorescence depends
on both the enzyme reaction and the degree of
deproteinization. Thus, the signals depend on buffer
substance and ionic strength as previously described
for a galactose-1-phosphate-uridyltransferase test
w27x. The solvent producing the highest AQ fluores-
cence, ethanolracetone, is also one of the best de-
proteinization agents. This mixture was selected for
all assay variants. Due to the low absorbances of
plasma Žcf. Table 2., moderate deproteinization is
sufficient here. With blood spots, low ionic strength
is used to achieve improved deproteinization. Never-
theless, absorbing material was not removed com-
pletely. To exclude false assessments, individual flu-
orescence intensities of AQ-standard solutions were
measured in parallel to enable normalisation of test
fluorescence in the presence of different residual
quenching compounds Žsee Section 4.4..
4.2. Biotinidase properties
The optimum assay pH, 6.5, is probably brought
about by the pH optimum range of biotinidase activ-
183
ity Ž5.0–5.5, data not shown; see also Ref. w31x., and
the fluorescence maximum of the product, AQ, above
pH 6.5.
The K m of serum biotinidase for the substrate
BAQ found with our miniaturized assay is higher
than found by Hayakawa et al. w23x but corresponds
closely to values found with Wastell’s assay w20x.
The dependence of enzyme stability on time and
temperature found with serum and with dried blood
spots is comparable to results of Hymes et al. w31x
and Pettit et al. w15x, respectively. The seemingly low
biotinidase activities of dried blood spots, which
amount to 3–4% of the corresponding serum activi-
ties Žcf. Table 4, Fig. 4. may be explained taking
into account differences of the assay temperatures,
hematocrit values, and cumulative activity reductions
upon drying, storage and transport. The difference of
the assay temperatures Ž14–208. and the hematocrit
values may explain an activity reduction to about
15%. Storage and transport Žsee Section 3.8. and
drying Žreduction to about 30%. are responsible for
the remaining activity decrease. To exclude sub-
strate-specific effects in the blood spot assay, the
biotinidase activities were measured in parallel using
two substrates. Blood spot biotinidase activities ob-
tained at 24 8C incubation temperature amount to
12.9 " 1.7% Ž n s 10. and 8.0 " 1.8% Ž n s 10. of
the values obtained with 4 ml whole blood at 38 8C
incubation temperature using BAQ and biotinyl-
aminobenzoic acid as substrate, respectively, indicat-
ing no significant substrate effects.
Taking into account the RQ10 value, incubation
time and temperature of the biotinidase assay may be
selected according to the laboratory requirements as
previously described for galactose-1-phosphate-
uridyltransferase activity measurements w27x. The in-
cubation of serum and dried blood spots at 38 8C for
2 and 6 h may be replaced, e.g. by incubation at
room temperature for about 6 and 17 h, respectively,
because fluorescence increase is linear with time
under these conditions Ždata not shown..
4.3. Imprecisions, sensitiÕity
Imprecisions of our standard assays with serum
and dried blood spots are in the range of impreci-
sions of other miniaturized serum assays w22,32x and
blood spot screening tests w15,27,29x, respectively.
184 E. Broda et al.r Clinica Chimica Acta 314 (2001) 175–185
Despite different definitions of sensitivity and
absolute activity ranges, the analytical sensitivity
calculated for our serum assay permits a reliable
detection of much lower biotinidase activities than
the assays reported in Refs. w29,32,33x, if the detec-
tion limits were compared with the corresponding
mean activity values.
For blood spots, to our knowledge, no other sensi-
tivity data have been reported. Our analytical sensi-
tivity is low enough to discriminate profound from
partial deficiency with properly handled specimens.
4.4. Standardisation
To exclude false negative assessment due to pos-
sible endogenous fluorescent compounds, we pro-
pose the parallel testing of individual blanks as
proposed for galactose-1-phosphate-uridyltransferase
measurement w27x. False positive assessment due to
preanalytical enzyme damage cannot be excluded;
however, false positive assessment due to the pres-
ence of quenching material could be prevented by
introduction of individual AQ fluorescence stan-
dards. Biotinidase activities can be calculated quanti-
tatively with both sample types using the two indi-
vidual AstandardsB, blanks and AQ-fluorescence. Due
to the decrease of biotinidase activity with drying
and increasing storage temperature, a seasonal de-
pendence of blood spot activity may be expected
w34x. Therefore, the cut-off as well as recall-rate and
sensitivity parameters used for blood spot screening
are strongly dependent on preanalytical conditions
Žclimate, seasonal and mailing conditions, and fre-
quency of preterm infants, too, which exhibit much
lower values. and must be adjusted along with the
measured daily mean activity of all samples as pro-
posed for galactosemia screening w27x.
4.5. Costs
Despite three-fold analysis of each sample Žindi-
vidual blank, AQ standard and biotinidase test., the
costs of our miniaturized assay with serum and dried
blood spots are in the range of 10 cents per sample
including reagents and plates. This is comparable
with the costs of screening using single determina-
tions w9,35x.
Acknowledgements
Critical reading of the manuscript and helpful
discussions by Dr. G.A. Cumme are gratefully ac-
knowledged.
References
w1x Hymes J, Wolf B. Biotinidase and its roles in biotin
metabolism. Clin Chim Acta 1996;255:1–11.
w2x Wolf B, Grier RE, Allen RJ, Goodman SI, Kein CL. Bio-
tinidase deficiency: enzymatic defect in late-onset multiple
carboxylase deficiency. Clin Chim Acta 1983;131:273–81.
w3x Wolf B, Grier RE, Secor McVoy JR, Heard GS. Biotinidase
deficiency: a novel vitamin recycling defect. J Inherited
Metab 1985;8:53–8.
w4x Wolf B, Heard GS, Jefferson LG, Proud VK, Nance WE,
Weissbecker KA. Clinical findings in four children with
biotinidase deficiency detected through a state-wide neonatal
screening program. N Engl J Med 1985;313:16–9.
w5x Wolf B, Heard GS, Weissbecker KA, Secor McVoy JR,
Grier RE, Leshner RT. Biotinidase deficiency: initial clinical
features and rapid diagnosis. Ann Neurol 1985;18:614–7.
w6x Salbert BA, Pellock JM, Wolf B. Characterization of seizures
associated with biotinidase deficiency. Neurology 1993;43:
1351–5.
w7x Thomason MJ, Lord J, Bain MD, et al. A systematic review
of evidence for the appropriateness of neonatal screening
programmes for inborn errors of metabolism. J Public Health
Med 1998;20:331–43.
w8x Lawler MG, Frederick DL, Rodriguez-Anza S, Wolf B, Levy
HL. Newborn screening for biotinidase deficiency: pilot study
and follow-up of identified cases. Screening 1992;1:37–47.
w9x Wolf B, Heard GS. Screening for biotinidase deficiency in
newborns: worldwide experience. Pediatrics 1990;85:512–7.
w10x Wolf B. Worldwide survey of neonatal screening for bio-
tinidase deficiency. J Inherent Metab Dis 1991;14:923–7.
w11x Strom CM, Levine EM. Chronic vaginal candidiasis respon-
sive to biotin therapy in a carrier of biotinidase deficiency.
Obstet Gynecol 1998;92:644–66.
w12x McVoy JR, Levy HL, Lawler M, et al. Partial biotinidase
deficiency: clinical and biochemical features. J Pediatr 1990;
116:78–83.
w13x Knappe J, Brummer
¨ W, Biederbick K. Reinigung und Eigen-
schaften der Biotinidase aus Schweinenieren und Lactobacil-
lus casei. Biochem Z 1963;338:599–613.
w14x Heard GS, McVoy JR, Wolf B. A screening method for
biotinidase deficiency in newborns. Clin Chem 1984;30:
125–7.
w15x Pettit DA, Amador PS, Wolf B. The quantitation of bio-
tinidase activity in dried blood spots using microtiter transfer
plates: identification of biotinidase-deficient and heterozy-
gous individuals. Anal Biochem 1989;179:371–4.
w16x Wolf B, Secor McVoy J. A sensitive radioassay for bio-
tinidase activity: deficient activity in tissues of serum bio-
 E. Broda et al.r Clinica Chimica Acta 314 (2001) 175–185
tinidase-deficient individuals. Clin Chim Acta 1983;135:
275–81.
w17x Evangelia L, Kakabakos SE, Evangelatos SA, Evangelatos
GP, Ithakissos DS. Determination of serum biotinidase activ-
ity with radioiodinated biotinylamide analogs. Methods En-
zymol 1997;279:442–51.
w18x Ahola T, Hemmila¨ I, Raunio R, Lovgren
¨ T. A time-resolved
fluorimetric method for measurement of biotinidase activity.
In: Schmidt BJ, editor. Current trends in infant screening.
Amsterdam: Excerpta Medica; 1989. p. 265–9.
w19x Kumasaka K, Muratsugu M, Fukui T, Kimura M, Takagi Y,
Hashizume N, et al. A new quantitative analytical method of
serum biotinidase activity using biocytin as a substrate and
its clinical significance in Japan. Clin Chim Acta 2001;306:
71–7.
w20x Wastell H, Dale G, Bartlett K. A sensitive fluorimetric rate
assay for biotinidase using a new derivative of biotin, bi-
otinyl-6-aminoquinoline. Anal Biochem 1984;140:69–73.
w21x Hayakawa K, Oizumi J. Determination of biotinidase activity
by liquid chromatography with fluorimetric detection. J
Chromatogr 1986;383:148–52.
w22x ¨
Pittkanen L, Tuuminen T. A quantitative fluorimetric mi-
cromethod used for neonatal screening of biotinidase defi-
ciency in Finland. Screening 1992;1:185–94.
w23x Hayakawa K, Yoshikawa K, Oizumi J, Yamauchi K. Deter-
mination of biotinidase activity with biotinyl-6-aminoquino-
line as substrate. Methods Enzymol 1997;279:435–42.
w24x Shein A, Skillen AW, Bartlett K. The application of fluori-
metric fast centrifugal analysis to the detection of biotinidase
deficiency. J Inherent Metab Dis 1987;10ŽSuppl. 1.:293–5.
w25x Rosenthal M.A., Simkins R.A., Akhaury R. Assay for en-
zyme activity using a microfluorimetric assay. International
application published under the patent cooperation treaty,
1995; Int Publ Number WO 95r33072.
View publication stats
185
w26x Suormala T, Ramaekers VT, Schweitzer S, et al. Biotinidase
K m -variants: detection and detailed biochemical investiga-
tions. J Inherent Metab Dis 1995;18:689–700.
w27x Rhode H, Elei E, Taube I, Podskarbi T, Horn A. Newborn
screening for galactosemia: ultramicro assay for galactose-1-
phosphate-uridyltransferase activity. Clin Chim Acta 1998;
274:71–87.
w28x Rhode H., Horn A., Scharff M., Wolfel¨ H. Mikrotiterplatte.
German application field 1997. Aktenzeichen 19739119.2.
w29x Yamaguchi A, Fukushi M, Arai O, et al. A simple method
for quantification of biotinidase activity in dried blood spot
and its application to screening of biotinidase deficiency.
Tohoku J Exp Med 1987;152:339–49.
w30x Rhode H., Horn A., Broda E. Verfahren zur empfindlichen
fluorimetrischen Messung der Freisetzung von 6-Amidoqui-
nolin. German application field 1999, Aktenzeichen
19930261.8.
w31x Hymes J, Fleischhauer K, Wolf B. Methods Enzymol 1997;
279:422–34.
w32x Weissbecker KA, Gruemer H, Heard GS, et al. An automated
procedure for measuring biotinidase activity in serum. Clin
Chem 1989;35:831–3.
w33x Forman DT, Bankson DD, Highsmith WE. Neonatal screen-
ing for biotinidase deficiency. Ann Clin Lab Sci 1992;22:
144–54.
w34x Thibodeau DL, Andrews W, Meyer J, Mitchell P, Wolf B.
Comparison of the effects of season and prematurity on the
enzymatic newborn screening tests for galactosemia and
biotinidase deficiency. Screening 1993;2:19–27.
w35x Schweitzer S, Sander J, Suormala T, Baumgartner R, Byrd
DJ, Brodehl J. Der Biotinidasemangel. Ergebnisse des
Neugeborenen-Screenings 1985–1989 in Niedersachsen.
Monatsschr Kinderheilkd 1991;139:349–54.