tremls in &alytical chemistry, vol. 1, no.

2, 1981 55

8 Barnes, R. M. (1978) CRC Crit. Rev. Anal. Gem. 7, 203 21 Boumans, P. W. J. M. and Bosveld, M. (1979) Spectrochim. Acta
9 Barnes, R. M. (1975) ICP inf Newsl. 1, 30 34B, 59
10 Greenfield, S. (1980) Analyst, 105, 1032 22 Parson, M. L., Forster, A. and Anderson, D. (1980) in An Atlas
11 Houk, R. S., Fassel, V. A., Fleach, G. D., Svec, H. J., Gray, A. of Spectral Znterferences in ICP Spectroscopy, Plenum Press, New
L. and Taylor, C. E. (1980) Anal. Chem. 52, 2283. York and London
12 Barnes, R. M. (ed.), (1976) in Emission Spectroscopy, Dowden, 23 Boumans, P. W. J. M. (1980) in Line Coincidence Tables for
Hutchinson and Ross, Stroudsburg, PA Inductively Coupled Plasma Atomic Emission Spectroscopy, Pergamon
13 Environmental Protection Agency (1980) Guidelines Establishing Press, Oxford and Elmsford, NY
Test Procedures for the Analysis of Pollutants;
Proposed Regulations,
Fed. Regist. 03 Dee 1979,44(233), 69424-69575; ZCPZnf Newsl.
1980,5, 528
14 Barnes, R. M. (ed.), in ICP Information Newsletter, University of Dr Ramon M. Barnes is a Profes-
Massachusetts, Amherst, MA 01003 sor of Chemistry at the University
15 Barnes, R. M. (ed.), (1978) in Applications of Inductively Coupled of Massachusetts, Amherst, MA
.Plasmas to Emission Spectroscopy, Franklin Institute Press, 01003, U.S.A. as well as being
Philadelphia editor and publisherof the ICP
16 Barnes, R. M. (ed.), (1979) in Applications of Plasma Emission Information Newsletter andchair-
Spectrochemtitry Heyden, Philadelphia man of the 1982 Winter Conference
17 Fuwa, K. and Haraguchi, H. (ed.), (1980) in Inductively Coupled on Plasma Spectrochemistry.
Plasma Emission Spectroscopy, Kagaku no Ryoiski, Zokan; 127, His research interests inclu&
235 pages, Tokyo both&damental and practical
18 Barnes, R. M. (ed.), (1981) in Developments in Atomic Plasma investigations of the inductively
Spectrochemical Analysis, Heyden, Philadelphia coupled plasma discharge, appli-
19 Environmental Protection Agency (1980) in Designation of cations ofplasma detectors in
Ambient Air Monitoring Equivalent Methods for Lead, Fed. Regist. chromatography, diagnostics of
06 Mar 1980,45(46), 14648-9; ICP Inf Newsl. 1980,6,49 spark discharges, and spectroscopic
20 Winge, R. K., Peterson, V. J. and Fassel, V. A. (1979) Appl. studies of low-pressure RF
Spectrosc. 33, 206 plasmas.

Clinical analysis of lipids using microbial

enzymes
The last decade has seen the increasing use of microbial
enzymes for clinical analysis. Glycerol oxidase, recently
discovered, has been applied to the analysis of serum
triglycerides.

T. Uwajima and 0. Terada The broad substrate specificity of the cholesterol

Tokyo, japan oxidase from B. sterolicum is of interest, when compared
The increased availability of microbial enzymes has with the narrower specificity of the membrane-bound
led to many applications in the area of clinical chem- cholesterol oxidase from Nocardia erythropolis596. Smith et
istry. These have been stimulated particularly by the al. reported7 that the Brevibacterium enzyme was
development of enzymatic methods for the determina-
.tion of serum cholesterol. Cholesterol oxidase (EC TABLE I. Substrate specificity of cholesterol oxidase
from Brevibacterium sterolicum
1.1.3.6) and cholesterol esterase (EC 3.1.1.13) were
found in the culture media of Brevibacterium sterolicum’
and PseudomonasJluorescens,2 respectively. Both enzymes Substrate Relative activity
are soluble and relatively thermostable. The crystal-
line cholesterol oxidase from B. sterolicum was shown3 to Cholesterol 100%
be a flavoprotein catalysing the oxidation, of 3B- Dehydro-epi-androsterone 41.4%
Pregnenolone 22.4%
hydroxyl groups of steroids such as cholesterol, 5-Androstene-3& 17B-diol 20.2%
dehydro-epi-androsterone, and pregnenolone (Table B-Sitosterol 19.7%
I). The cholesterol esterase from P.jZuorescens catalyses B-Cholestanol 13.2%
the hydrolysis of long-chain fatty acid esters of Stigmasterol 10.0%
cholesterol4. Reagent kits containing both cholesterol Ergosterol 0
Digitoxigenin 0
esterase and cholesterol oxidase have been successfully Estradiol 0
developed for the assay of total serum cholesterol in Diosgenin 0
routine clinical analysis - it being significant for the Cholic acid 0
assessment of many clinical conditions, such as
arteriosclerosis and for monitoring the risk of thrombo- The enzyme activity was determined by estimating the initial
sis and myocardial infarction. rate of oxygen uptake manometrically.
0 165~9!336/81/0000-oooO/SO2.50 o 1981
Elscvicr
Scientific Publishing Company
56 tremis in analytizal chemistry, vol. I, io. 2, 1981

capable of catalysing the oxidation of 5-androsten-3fi- CoA oxidase. The level of free fatty acids in human
01s containing as many as three additional hydroxyl blood is of importance for the diagnosis of diabetes and
groups. The enzyme has accordingly been found applic- other disorders including thyroid dysfunction.
able to the characterization of dihydroxy- and tri-
hydroxy-5-androsten- 17-ones in human amniotic fluid A new copper-haemoprotein -glycerol
using gas-liquid chromatography-mass spectrometry oxidase
(GC-MS). Gaskell et al. also applied8 the Breuibacterium In the course of an investigation on glycerol metab-
enzyme to the preparation of deuterium-labelled olism by micro-organisms, glycerol oxidase, a novel
testosterone, suitable for use as an internal standard in glycerol-oxidizing enzyme, was found in some strains
the quantification of testosterone by GC-MS. of Aspergillus, Neurospora, and Penicillium’7. Glycerol
oxidase catalyses the oxidation of glycerol in the
Analysis of bile acids presence of oxygen to form glyceraldehyde and hydro-
Sar-Hydroxysteroid dehydrogenase (EC 1.1.1.50) is gen peroxide, without the need for any exogeneous
a well-known enzyme capable of pyridine nucleotide- cofactors (see scheme 3). The purified enzyme from
linked, reversible interconversions of 3@-hydroxyl and Aspergillus japonicus is homogeneous on ultracentrifuga-
ketone groups of steroid@. A crystalline 3~ tion and acrylamide-gel electrophoresisia. It has a mol.
hydroxysteroid dehydrogenase was prepared in the wt of 400,000 and an isoelectric point of 4.9. The
presence of polyethylene glycol from the cell extract of enzyme shows the typical absorption spectra of a
Pseudomonas putida 10. The enzyme is specific for 3~ haemoprotein; the absorption maxima are located at
hydroxysteroids with A:B cis fusion, e.g. cholic acid, 557 and 430 nm in the reduced form, and at 557,530,
deoxycholic acid, and taurocholic acidii. The sub- 420, 280 and 238 nm in the oxidized form. Anaerobic
strate specificity indicates that the enzyme can be used addition of glycerol to the enzyme causes both a shift of
for the analysis of free and conjugated bile acids in the Soret band from 420 to 410 nm and a decrease in
serum. The estimation of total serum bile acids is absorbance at 557 and 530 nm (Fig. 1). The pyridine
important for the diagnosis of hepatobiliary disorders, ferrohaemochrome of the enzyme shows an almost
many of which are associated with elevated concentra- identical absorption spectrum to that of haematin,
tions of these metabolites in the blood. indicating that the iron porphyrin in the enzyme is
protohaem IX. The enzyme contains 0.94 mol. of
haem per enzyme protein mol., as determined by the
Determination of phospholipids
extinction coefficient of the pyridine ferrohaemo-
The phospholipid levels in plasma reflect the
chrome (E = 3.2 X IO-2 at 557 nm) 19. Sodium azide
function of the liver. Serum contains a great variety of
and hydroxylamine are potent competitive inhibitors
phospholipids, but more than 90% of the total is com-
with Ki values of 2.0 and 1.2 ~1,respectively. On the
prised by the lecithins, sphingomyelins, and lyso-
other hand, analysis of the enzyme by atomic absorp-
lecithins. In the analysis of serum phospholipids,
tion spectrophotometry reveals the presence of copper
hydrolysis catalysed by phospholipase D (EC 3.1.4.4)
ions at a concentration of 2.04 gram atoms per enzyme
releases choline, which can be estimated via the action
of choline oxidase. Choline oxidases (EC 1.1.3.17)
have been found in Cylindrocarpon didynumlz, Arthro-
batter globifoormi.+, and Brevibacterium album’*, and
420
characterized as flavoproteins in the form of purified
preparations. These enzymes can catalyse the oxida-
tions of both choline and betaine aldehyde using
molecular oxygen as a primary electron acceptor and
producing hydrogen peroxide.

Assay of free fatty acids

Acyl-CoA oxidase was obtained in crystalline form
from the cell extract of an n-alkane-utilizing yeast,
Candida tropicalis 15. The enzyme was shown to be
flavoprotein which was active toward acyl-CdA’s with
carbon chain length of 4-20. The oxidation of acyl-
CoA by the enzyme proceeds as follows:
600 600

Acyl-CoA + 0s w Enoyl-CoA + H202 wave length, nm

(1)
For the estimation of serum free fatty acids, Shimizu
et al.16 developed an enzymatic method which was
based on the activation of free fatty acids by acyl-CoA
synthetase (EC 6.2.1.3), followed by the action of acyl-
Fig. I. Absorption Spectra of Glycerol Oxidasc; 3.8 nmol in 0.5 ml of 0.02 M
N-Tris(hydroxymethyl)-methyl-2-amino&an sulfonic acid (TES)-
NHeOH buff,, pH 7.0. A, thefewic form; B, ajkr the addition of glycerol
(10 ~1) to A unak anaerobicconditions; C, thef~ousjiwm obtained by the
addition of sodium dithionitc (5 ~1) to A unak anaerobic conditions.
trenak in a;laIyticaEchcmist~, vol. 1, no. 2,198l 57
.

dye in the sequence of the following reactions:

Triglyceride + HZ0 Lipoprotci”lipas Glycerol +

3 Fatty acids (2)

Glycerol + 0s G’ycero’Oxidase+Glyceraldehyde +
H202 (3)
4-Aminoantipyrine + N2 Ethyl-N2-( 3 methylphenyl)
Nl acetylethylenediamine
Peroxidase + 2H202 (4)
(EC 1.11.1.7)
9 Quinoneimine dye + 4 H20
The reaction mixture containing 20 pl serum in a final
volume of 3.0 ml is incubated at 37°C for 10 min, and
the resulting dye is read at 555 nm on a spectrophoto-
Fig. 2. ESR Spectra of Glycerol Oxidasc; 1.8 nmol in 0.5 ml of 0.02 M meter. The enzymatic method is more simple, sensi-
TES-NH,OH buffer, PH 7.0. A, the enqme only; B, C and D, a&r the tive, and far less susceptible to interference from
additions ofglyccrol(10 pmol), sodium dithionitc (5 pnol) anddiethyldithio- bilirubin, ethylenediaminetetra-acetic acid, and oxalic
carbamatc (5 pmol), respectively to A under anaerobic conditions. The acid, than chemical methodssl-2s.
spectra were recordedat a microwavepower of 4 m W, a modulation akplitudc
of 5 gauss, a field of 3500 + 100 G, 9.19 GHz, and at a tmrpcrature of
- 180%. Conclusions
Microbial enzymes are becoming increasingly
important in the clinical estimation of serum lipids
such as cholesterol, triglycerides, free fatty acids,
protein mol. Electron spin resonance signals are and phospholipids. This trend is not only confined to
observed at g= 1.99, g=2.00 and gz2.02. The g= 1.99 the analysis of serum constituents, but is also apparent
and g=2.02 signals are diminished by the anaerobic in the field of diagnostic enzymology; for example, in
addition of glycerol, and the three signals disappear the determination of glutamic oxalacetic transaminase
completely on the addition of either a reducing agent and glutamic pyruvic transaminase using pyruvate
(dithionite) or a copper chelating agent (diethyldithio- oxidase. Adoption of enzymatic assays is growing as a
carbamate), as shown in Fig. 2. These results indicate result of their precision, specificity, and the commer-
that the protohaem IX and cupric copper ions associ- cial availability of purified enzyme preparations.
ated with the enzyme are directly involved in the Enzymological investigations on glycerol degrada-
electron transfer from the substrate. tion by micro-organisms have resulted in the discovery

TABLE II. Substrate specificity of glycerol oxidase

Analysis of triglycerides from Asprgillus japonicus
The determination of serum triglyceride concen-
trations has been a common and significant assay
in clinical laboratories, since the triglycerides were Substrate Relative activity
identified as risk factors in coronary artery disease.
Glycerol 100%
Accurate analysis of serum triglycerides, as well as that Dihydroxyacetone 58.90%
of serum cholesterol, is now required for adequate 1,3-Propanediol 6.73%
diagnosis of hyperlipidaemia. 1,3-Butanediol 6.19%
The substrate specificity of glycerol oxidase from A. 1,4-Butanediol 1.19%
Glycerol 3-phosphate 0
japonicus was investigated with a variety of hydroxylic
Dihydroxyacetone phosphate 0
compounds (Table II). Glycerol is oxidized most D-Galactose 1.46%
rapidly and dihydroxyacetone is also oxidized at a D-Fructose 0.47%
significant rate: however, glycerol 3-phosphate, meth- .D-Glucose 0
anol, ethanol, and polyvinyl alcohol are not oxidized at Saccharose 0
Methylalcohol 0
all. Among the saccharides, galactose and fructose are
Ethylalcohol 0
slightly oxidized. The pH optimum for the oxidation of Polyethylene glycol 0
glycerol is 7.0. The K, value and I’,,,,, are 10.4 mm and
935.6 pmol min-rmg-1, respectively. Accordingly,
glycerol oxidase, in combination with lipoprotein The enzyme activity was determined by measuring hydrogen
peroxide formed spectrophotometrically. The assay method is
lipase (EC 3.1.1.3), can be employed for the determi-
based on oxidative coupling of the hydrogen peroxide with 4-
nation of serum triglycerides. The enzymatic assay aminoantipyrine and phenol in the presence of peroxidase, and for-
method is based on the formation of a quinoneimine mation of a chromogen with maximum absorption at 500 nm*O.
58
of a previously unknown enzyme, glycerol oxidase,
capable of the specific oxidation .of glycerol. This
enzyme is a unique copper-haemoprotein which cat-
alyses two-electron reductions of oxygen, with one
molecule of hydrogen peroxide formed per molecule of
oxygen consumed. It is expected that, together with
further studies on the metabolism of serum constitu-
ents and diagnostic enzymology, the isolation of other
novel microbial enzymes will lead to the development
of more reliable and sophisticated methods for clinical
analysis.
Acknowledgements
We thank Prof. C. J. W. Brooks (Glasgow Uni-
versity) for his valuable advice on the manuscript. We
are also grateful to Dr T. Suzuki, Director of our re-
search laboratory, for his encouragement in the com-
pilation of this review.
trends in analytical chemistryvol. I, io. 2, 1981
.
References
1 Uwajima, T., Yagi, H., Nakamura, S. and Terada, 0. (1973)
Agric. Biol. Chem. 37, 2345
2 Uwajima, T. and Terada, 0. (1975) Agric. Biol. Gem. 39, 1511
3 Uwajima, T., Yagi, H. and Terada, 0. (1974) Agrix. Biol. Chem.
38, 1149
4 Uwajima, T. and Terada, 0. (1976) Agric. Biol. Gem. 40, 1957
5 Smith, A. G. and Brooks, C. J. W. (1975)J. Chromatogr. 112,499
6 Smith, A. G. and Brooks, C. J. W. (1976) J. Steroid Biochcm. 7,
705
7 Smith, A. G., Joannou, G. E., Mtrk, M., Uwajima, T., Terada,
0. and Brooks, C. J. W. (1978) J. Chromatogr. 152, 467
8 Gaskell, S. J. and Finlay, E. M. H. (1980) J. Labelled Compounds
and Radiopharmaceuticals, 17, 86 1
9 Talalay, P. and Marcus, P. I. (1956) J. Biol. Chem. 218, 675
10 Uwajima, T. and Terada, 0. (1978) Agric. Biol. Gem. 42, 1577
11 Uwajima, T. and Terada, 0. (1979) Agric. Biol. Chem. 43, 1521
12 Tani, Y., Mori, N. and Ogata, K. (1979) Agric. Biol. Chem. 41
1101
13 Ikuta, S., Imanura, S., Misaki, M. and Horiuchi, Y. (1977) J.
Biochem. 82, 1741
14 Nakanishi, T., Shigemasa, Y. and Kawamoto, I. (unpublished
data)
15 Shimizu, S., Yasui, K., Tani, Y. and Yamada, H. (1979)
Biochem. Biophys. Res. Commun. 91, 108
16 Shimizu, S., Tani, Y., Yamada, H., Tabata, M. and Murachi,
T. (1980) Anal. Biochem. 107, 193
17 Uwajima, T., Akita, H., Ito, K., Mihara, A., Aisaka, K. and
Terada, 0. (1980) Agric. Biol. Gem. 44, 399
18 Uwajima, T. and Terada, 0. (1980) Agric. Biol. Chem. 44,2039
19 Paul, K. G., Theorell, H. and Akeson, A. (1953) Acta Chem.
&and. 7, 1248
20 Allain, C. C., Poon, L. S.,.Chan, C. S. G., Richmond, W. and
Fu, P. C. (1974) Clin. Gem. 20, 470
21 Randrup, A. (1960) &and. J. Clin. Lab. 12, 1
22 Van Handel, E. (1961) Clin. Gem. 7, 249
23 Fletcher, M. J. (1968) Clin. Chim. Acta 22, 393
Dr. Takayuki Uwajima graduated
.f?om the Fame&vofAgriculture,
Kpto University in RX.5 and
studied eny*nc chemistry as a
jrostgraduate at the university
under the guidance of Professor
Hidcaki Yamadajom 1965until
1967. Subsequently, he was
employedby the Kyowa Hakko Co.,
andnow works in th Ev
Department of their Tokyo
Research Laboratory Asahi-ko 3-
6-6, Tokyo 194, Japan. He
received his PhDjom Kyotot
University in 1978.
Dr. Osamu Terada graduated
jam the Facula ofAgriculture,
Tokyo University, in 1951. He was
employed by the Kyowa Hakko
Co., in 1951 and has worked in
their Tokp Research Laboratory
for3Oyears. Tokyo University
awar&d him a PhD in 1!X1. Dr.
Terada has many interests in the
field of applied-microbiology. He
became the managing director of
Kyowa Medex Co., Tokyo in
1981.