4. Lowe DA. A guide to international recommendations on names and symbols for
quantities and on units of measurement.
Geneva: WHO, 1975, 314 pp.
5. Council of Biology Editors Style Manual,
5th ed. Bethesda, MI) 20814: Council of
Biology Editors, Inc., 1983.
6. PowsnerER, Anido G, Armbrecht B, et
al. Quantities and units. Committee report,
vol 3, no. 3. Villanova, PA 19085: NCCLS.
Table 1. Effect of Sample/Reagent Volume Ratio on NAG Activity
Measured by Various Methods
Method I
MethodII
Method III
Sample/reagent
RelatIveactIvity,
%,
mean
(and
SD)
volumeratIo
1/3
86( g)b
93(10)
40(17)#{176}
1/6
100 ( 8)
102 ( 7)
63
1/16
103( 6)
107 (11)
89(20)
1/25
104 (13)
98 ( 7)
99 (12)
Relativeto the activity measured in gel-filteredsamples (n
20): for Method I,thisamounted to a
mean (andSD) of 31.2(21.7) U/L;forMethod II, 12.7(11.9)U/L;andfor MethodIll, 12.3 (10.4) U/L.
bsignificantlydifferent(pairedf-test)from NAG activitymeasuredingel-filteredsamples, by at least P
(14)t1
=
More about DetermInation of NAcetyl-$-o-glucosaminldase
Activity
In UrIne wIthout Pretreatment of
Samples
To the Editor:
4-Nitrophenyl-N-acetyl--I)-glucosaminide is apparently
the most widely
used substrate for determination
of
urinary N-acetyl-3-n-glucosaminidase
(NAG; 2-acetamido-2-deoxy-3-r-glucoside acetamidodeoxyglucohydrolase,
EC 3.2.1.30) activity. Because NAG
activity is affected by inhibitors present in urine, gel filtration or dilution
of urine samples is recommended to
minimize their influence (1). However,
because the inhibitors
interact with
the enzymatic reaction competitively,
we considered that the inhibition
could
also be diminished by optimizing the
substrate concentration.
We re-investigated,
therefore, the reaction conditions of NAG determination at 37#{176}C
with the above substrate
and found final concentrations of, per
liter, 8 mmol of 4-nitrophenyl-N-acetyl-p-r-glucosaminide, and 25 minol of
acetate buffer (pH 4.20) to be optimum
(2). This concentration, which is higher
than recommended by others (3, 4),
amounts to about 10 Km (2,3).
Using these reaction conditions
(Method I), we measured urinary NAG
activities in 10 healthy adults and 10
renal-transplant recipients at various
urine sample/reagent
ratios, set the
values measured in samples after gel
filtration (3) at 100%, and calculated
the percentages of the activity at the
different ratios (Table 1).
We compared these results with
those obtained with the two recently
introduced new methods, in which 3cresolsulfonphthaleinyl
(5) and w-nitrostyryl derivatives (6) of N-acetyl-/3n-glucosaminide
are the substrates
(Methods II and ifi, respectively) (Table 1). For Method U, we used a test kit
from Boehringer Mannheim GrnBH,
Mannheim, F.R.G., and measured the
samples at the various sample/reagent
ratios at identical final substrate concentrations as prescribed for the original test. For Method ifi, we replaced
<0.05.
the stopping buffer of sodium carbonate/bicarbonate by 1.0 moIfL of Tris
buffer (pH 10.0) because the released 2methoxy-4-(2’-nitrovinyl)-phenol
product is more stable in this solution.
Our results (Table 1) prove that the
conventional test, with 4-mtrophenylN-acetyl-f3-n-glucosaminide at an optimal substrate concentration, provides
sufficiently reliable results without gel
ifitration, at sample/reagent volume
ratios of 1/6. We recommend the use of
this substrate-optimized method rather than determinations with suboptimal concentrations and higher sample/
reagent ratios [e.g., 1/51 (1) or 1/19(5)],
which require longer incubation times
or reduce the analytical sensitivity of
the method. Although 4-nitrophenol
has a lower molar absorptivity (18.6 L
#{149}
mol1 cm’ at 405 nm) than the
3-cresolsulfonphthalein
(40.67
L mol’
cm’ at 580 nm) released in
Method for the 2-methoxy-4-(2’-nitrovinyl)phenol (26.8 L mol’
cm’ at
505 nm) in Method ifi, the 4-nitrophenol technique does not need any
longer reaction times than the other
methods. That is because the lower
molar absorptivity
is compensated by
the preferential reactivity of NAG with
this substrate (see absolute activities
given in footnote a
Table 1). Consequently, the sole disadvantage of using
4-mtrophenyl- N -acetyl- B -n-glucosaminide, as compared with 3-cresol sul-
2. Jung K, Nesener E. Bestimmung der NAcetyl--D-glucosaminidase-Aktivitat
im
Urin mit einemkolorirnetrischen mid fluorimetrischen
Test. Z Med Labor-Diagn, in
press.
3. Maruhn D. Rapidcolorimetricassayofgalactosidaseand N-acetyl-fl-o-glucoeaminidase in human urine. Chin Chim Acta
1976;73:453-61.
4. Horak E, Hopfer SM, Sunderman Jr FW.
Spectrophotometric assay for urinary Nacetyl--D-glucosaminidase activity. Clin
Chem 1981;27:1180-5.
5. Goren MP, Wright RK, Osborne S. Two
automated procedures for N-acetyl-p-s-glucosaminidase determination evaluated for
detection
of drug-induced tubular
nephrotoxicity. Clin Chem 1986;32:2052-5.
6. Yuen CT, Kind PEN, Price RO, Praill
PFG, Richardson AC. Colorimetric assayfor
N-acetyl-p-i-glucosamimdase
(NAG) in
pathological urine using the w.nitrostyryl
substrate: the developmentof a kit and the
comparison of manual procedure with the
automated fluorunetric method. Ann Chin
Biochem 1984;21:295-300.
-
fonphthaleinyl-N-acetyl-B-n-glu-
cosaminide, for example, seems to be
that urine sample blanks are necessary. It is, however, an inexpensive
and generally available substrate, offering a cost-effective method:
We gratefully thank Dr. R. G. Price
(King’s College, University London,U.K.)
for providing w-nitrostyryl-N-acetyl--sglucosaminide.
References
1. 1Am CW. N-Acetyl--s-gIucosaminidaae
assay by the p-nitrophenol technique: inhibitory effects of urine decreased by gel
ifitration and simple dilution [Tech Briefi.
Clin Chem 1987;33:713-4.
1002 CLINICAL CHEMISTRY, Vol. 34, No. 5, 1988
Klaus Jung
Silke Klotzek
Dept. of Exptl. Organ Transplantation
Univ. Hosp. Charit
Humboldt University Berlin
Leninal lee 49
DDR -1017 Berlin, GI1R.
Quantification of Glycosylated
Hemoglobin by Binding of Inositoi
Hexaphosphate
To the Editor:
Development of a simple, rapid assay for glycosylated hemoglobin (Glib)
is an important prerequisite
if results
are to be made available within the
time frame of a diabetic outpatient
clinic.
In 1980, Moore et al. (1) published an
abstract that stated that Glib could be
quantified by measuring
changes in
the spectrum
at 560 and 633 nm
caused by binding of inositol hexaphosphate (phytic acid). No description of
the methodology,
however, was ever
subsequently published. In 1982, Walinder et al. (2) evaluated a kit (“Glycospec”; Abbott Laboratories)
based on
the original work of Moore et al. (1).
From that initial study (2) it was concluded that the method had particular
advantages in its rapidity and ease of
automation,
although potential problems existed with respect to standards
and calibration. The procedure has
since been cited by several major reviews of protein glycosylation
(3, 4)
with the distinct inference that it represents a viable assay for the quantification of GHb.
Our attempts to reproduce the method, using a variety of conditions, all
proved unsuccessful. The spectral
changes are very small and are quite
sensitive to pH; the reader is referred
to the paper by Maliyakal and Waterman (5). Recent correspondence with
one of the authors of the “kit-evaluation” study revealed that they have not
subsequently pursued the method because of major problems with standards and an inability to reproducibly
measure the finite changes in abeorbance. it is our understanding that the
Glyospec kit is no longer available and
a search of the literature
revealed no
other references to this assay other
than the two already cited (1, 2). The
methodology
originally
described by
Moore et al. (1) should therefore be
viewed in light of the preceding information.
dration,
and metabolic
acidosis
(NaHCO3 8.7 mmol/L), Because no precise information was available concerning dietary status or medication,
an organic aciduria was suspected and
rapid screening for an inherited metabolic disease was achieved by NMR at
80 MHz.
A ‘H NMR spectrum was recorded at
80 MHz on a WP 80 WG spectrometer
(Bruker, Karlsruhe F.R.G.), operating
in the pulse Fourier-transform
mode
with a quadrupole detection. The urine
sample was lyophilized, then dissolved
in 2H2O containing 3-(trimethylsilyl)-
Saiicyiate Poisoning Detected by ‘H
NMR Spectroscopy
To the Editor:
In a recent paper (1), iles et al.
recommended the use of proton nuclear
magnetic resonance (if NMR) spectroscopy for the detection and the study
of organic acidurias. We have used this
method to investigate a urine sample
obtained from a four-month-old girl.
This patient presented with the following symptoms: agitation,
polypnea,
hyperexcitation, fever (39.9 #{176}C),
dehy-
111
a)
III
II
50
7.5
7.0
5.5
1
coo.’
H,
143
ppm
er
b)
References
1 Moore EG, Stroupe SD, Blecka LI. Automated spectrophotornetric assayfor glycosyhated hemoglobin. Diabetes 1980;29 (Suppl
2):70A.
2. Walinder 0, Ronquist G, Fager P-J. New
spectrophotometric method for the determination of hemoglobin A1 compared with a
microcolumn technique.
1982;28:96-9.
5.0
1.5
1.0
CO-NH-CH,-COOH
C)
HroH
HH
H
Chin Chem
3. Mayer TK, Freedman ZR. Protein glycosylation in diabetes mellitus: a review of
laboratory measurements and of their clinical utility. Clin Chim Acta 1983;127:14784.
4. Bernstein RE. Nonenzyinatically glycosylated proteins [Review]. Adv Chin Chem
198726:1-78.
5 Maliyakal EJ, Waterman MR. Differential effects of pH and inositol hexaphosphate
on the spectroscopicproperties of the alpha
and beta subunits in methemoglobins M
Milwaukee and A. Biochim BiophysActs
II
SO
7.5
7.0
H.
#{149}.8
H.
coo.’
III
1979;578:269-80.
..
George Phillipou
Chris L Seaborn
Endocrine & Diabetes Lab.
The Queen Elizabeth Hosp.
Wood yule, South Australia 5011
.
Fig. 1. Ammatic region of 1H NMR spectra of urine samplefrom the patient (a) and of
the pure metabolites of acetylsahcylic
acid:2-hydroxybenzolc acid (b), o.hydroxyhippunc acid (, and 2,5-dihydroxybenzolc acid (
Partsb-d. runat 80 MHz. are In agreementwiththoserun at 80 MHz, In The Aldrichlibraryof
NMRspectra.J Pouthert JR Can,belI, eds. Milwaukee:AldrichChemicalCo., Inc.
CLINICALCHEMISTRY, Vol. 34, No. 5, 1988 1003