Method and composition for determination of glycerol

A method for determination of glycerol by using a reagent system comprising glycerol dehydrogenase and a pyridine nucleotide coenzyme, characterized in that an enzyme selected from the group consisting of triokinase and dihydroxyacetone kinase is incorporated into the reagent system to eliminate the formed dihydroxyacetone or D-glyceraldehyde in the assay system.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to a method and composition for determination 
of glycerol, and particularly to a method and composition for 
determination of glycerol by using glycerol dehydrogenase, characterized 
in that the glycerol dehydrogenase-catalyzed reaction for the 
determination is promoted by dihydroxyacetone kinase or triokinase. The 
invention is specially useful for the determination of glycerol and 
triglyceride be contained within biological fluids. 
2. Description of the Prior Art 
The determination of glycerol and triglyceride contained within blood is 
very important for diagnosis of hyperlipemia. In particular, hypercontent 
of triglyceride in blood is characteristic of arteriosclerosis, coronary 
insufficiency, myocardial infarction, etc. For early diagnoses or 
treatments of these diseases, a rapid and accurate method is desired for 
determination of triglyceride content in blood. 
A method for determination of triglyceride using lipase and glycerol 
dehydrogenase is characterized by the following reaction sequence. 
##STR1## 
NAD(P).sup.+ represents nicotinamide adenine dinucleotide (phosphate), 
NAD(P)H represents reduced NAD(P).sup.+, and GDH represents glycerol 
dehydrogenase. 
First, triglyceride is hydrolyzed into glycerol and fatty acids by the 
action of lipase, then the glycerol is oxidized into dihydroxyacetone (or 
D-glyceraldehyde) in the presence of NAD(P).sup.+ by the action of 
glycerol dehydrogenase. The formed NAD(P)H is measured by 
spectrophotometrical or fluorometrical assay, thereby determining glycerol 
and consequently triglyceride. 
The equilibrium of the above GDH-catalyzed reaction is shifted in the 
reverse direction. Thus, the reaction does not sufficiently proceed in the 
forward direction. In addition, it takes a long time to attain 
equilibrium. Consequently, the measurement is unsatisfactory in precision 
or sensitivity, and the range of measurable glycerol or triglyceride 
concen- trations. 
In order to overcome the above difficulty, it was necessary to carry out 
the reaction in as high pH as from 10 to 11, or to add excess NAD(P).sup.+ 
to the reaction mixture. 
A possible preferable approach to the promotion of the above GDH-catalyzed 
reaction is to eliminate the product dihydroxyacetone or D-glyceraldehyde 
from the assay system. For example, the method of converting the 
dihydroxyacetone into the corresponding hydrazone by adding hydrazine was 
attempted to promote the GDH-catalyzed reaction [Rinshobyori (Clinical 
Pathology), 24, 855 (1976)]. However, this method is unsatisfactory, since 
the enzyme is inactivated by hydrazine during the reaction. 
SUMMARY OF THE INVENTION 
An object of the invention is to provide a method for determination of 
glycerol which can overcome the above-mentioned problems. 
Thus, an object of the invention is to provide a method for determination 
of glycerol by using GDH, wherein the reaction can be effectively promoted 
under the conditions favorable for enzymes. 
Another object of the invention is to provide such a reagent composition 
for determination of glycerol by using GDH that is effective for promoting 
the GDH-catalyzed reaction under favorable conditions for enzymes. 
According to the present invention, there is provided a method for 
determination of glycerol by using a reagent system comprising GDH and a 
pyridine nucleotide coenzyme, characterized in that an enzyme selected 
from the group of consisting of triokinase and dihydroxyacetone kinase is 
incorporated into the reagent system to eliminate the formed 
dihydroxyacetone or D-glyceraldehyde in the assay system. 
According to the present invention, there is provided a composition for 
determination of glycerol which comprises (1) an enzyme selected from 
triokinase and dihydroxyacetone kinase, (2) glycerol dehydrogenase, (3) a 
pyridine nucleotide coenzyme, (4) a phosphate donor, and (5) a divalent 
metal cation.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
Triokinase (ATP: D-glyceraldehyde-3-phosphotransferase EC 2.7.1.28) is an 
enzyme which catalyzes the reaction of transferring the phosphate group of 
a phosphate donor such as adenosine-5'-triphosphate (referred to as ATP) 
to D-glyceraldehyde as shown by the following equation: 
EQU D-glyceraldehyde+ATP.fwdarw.D-glyceraldehyde-3-phosphate+ADP 
Triokinase is known to catalyze the phospholylation of dihydroxyacetone 
(referred to as DHA) and D-glyceraldehyde at nearly the same rate. 
Dihydroxyacetone kinase (referred to as DHAK) is an enzyme which catalyzes 
the reaction of transferring the phosphate group of phosphate donor such 
as ATP to dihydroxyactone but acts only slightly on D-glyceraldehyde and 
therefore is regarded to be different from triokinase. The reaction of 
dihydroxyacetone kinase is represented by the following equation: 
EQU Dihydroxyacetone+ATP.fwdarw.Dihydroxyacetone phosphate+ADP 
Triokinase is known to exist in guinea pig liver, rat liver, and Bacillus 
subtilis [Meth. Enzymol. 5, 362 (1962); Eur. J. Biochem., 31, 59 (1972), 
and The Enzyme, 2nd ed., 6, 75 (1962)]. Dihydroxyacetone kinase has been 
found in Candida methylica [Z. Allg. Mikorobial., 389 (1980) and ibid., 
21, 219 (1981)], Gluconobacter suboxydans (Joint Technical Meeting held by 
the Chubu Blanch and the Kansai Blanch of the Agricultural Chemical 
Society of Japan, Oct. 9, 1981, Abstract of the lectures, page 3), 
Acetobacter xylinum (J. Bacteriol., 127, 747 (1976)], Dunaliella, a green 
alga, [Plant Physiol., 59, 15 (1977), and Biochim. Biophys. Acta, 615, 1 
(1980)]. The present inventors newly found that dihydroxyacetone kinase is 
abundantly produced by the strains of genus Schizosaccharomyces. 
All the above cited triokinases and dihydroxyacetone kinases can be used 
for the invention, the dihydroxyacetone kinase produced by genus 
Schizosaccharomyces is particularly preferable for its productivity and 
properties. 
Available dihydroxyacetone kinase-producing strains of genus 
Schizosaccharomyces, include, for example, S. pombe IFO 0340 and IFO 0354, 
S. malidevorans IFO 1608, S. japonicus IFO 1609, and S. octosporus IAM 
12257. Among these, S. pombe IFO 0354 is preferred for its high 
productivity. All the strains above-mentioned have been deposited in 
recognized depositories respectively, abbreviated mark of "IFO" therein 
representing Institute for Fermentation, Osaka, Japan, and "IAM" 
representing Institute of Applied Microbiology, University of Tokyo, 
Japan. 
Dihydroxyacetone kinase produced by S. pombe IFO 0354 comprises two 
isoenzymes (referred to as DHAK(I) and DHAK(II), properties of them are as 
follows: 
(1) Reaction: The enzymes catalyze the reaction of transferring the 
phosphate group of a phosphate donor such as ATP to DHA to form 
dihydroxyacetone phosphate. 
(2) Substrate specificity: The enzymes act on dihydroxyacetone, but 
slightly on DL-glyceraldehyde, and not on glycerol, 1,2-propanediol, 
1,3-propanediol, acetol, acetoin, glycerol-3- phosphate, or DL-glyceric 
acid. 
(3) Specificity of the enzymes for phosphate donor:ATP is best phosphate 
donor and uridine-5'-triphosphate slightly acts as a phosphate donor for 
both DHAK(I) and DHAK(II). Inosine-5'-triphosphate, 
cytidine-5'-triphosphate, and guanosine-5'-triphosphate are inert. 
(4) Specificity for divalent metal cation: The enzymes exhibit no activity 
in the absence of divalent metal cation such as Mg.sup.2+, Ca.sup.2+, 
Co.sup.2+, or Mn.sup.2+. DHAK(I) exhibits the maximum activity in the 
presence of Ca.sup.2+ and DHAK(II) in the presence of Mg.sup.2+. 
(5) Optimum pH: Approximately 7.3 for both DHAK(I) and DHAK (II). 
(6) pH Stability: DHAK(I) is stable in a pH range of from 5 to 11, and 
DHAK(II) from 6 to 11. 
(7) Optimum temperature: 
The optimum temperature is about 60.degree. C. for DHAK(I) and about 
55.degree. C. for DHAK(II). 
(8) Thermal stability: DHAK(I) is stable below 50.degree. C. and DHAK(II) 
below 40.degree. C. 
(9) Km values: Michaelis constants (Km values) for dihydroxyacetone, 
DL-glyceraldehyde, and ATP under the reaction conditions of at pH 7.5 and 
at 25.degree. C. are as follows: 
DHAK(I): 8.4.times.10.sup.-6 M, 2.1.times.10.sup.-5 M, 2.2.times.10.sup.-4 
M 
DHAK(II): 2.0.times.10.sup.-5 M, 3.2.times.10.sup.-5 M, 9.1.times.10.sup.-4 
M 
DHAK(I) and DHAK(II) exhibit the sufficient activities at 4 mM or above 
concentration of Mg.sup.2+. 
(10) Molecular weight: Molecular weights of DHAK(I) and DHAK(II), as 
measured by gel filtration method with Sephadex G-200 (Pharmacia Fine 
Chemicals Co.), were both calculated to be about 145,000. 
Preferred embodiments are described below of the method for determination 
of glycerol according to the invention. First, glycerol is dehydrogenated 
into dihydroxyacetone or D-glyceraldehyde by the action of GDH in the 
presence of NAD(P).sup.+. The formed dihydroxyacetone or D-glyceraldehyde 
is converted into dihydroxyacetone phosphate or 
D-glyceraldehyde-3phosphate by the action of triokinase or 
dihydroxyacetone kinase to eliminate dihydroxyacetone or diglyceraldehyde 
from the assay system. Thus. the GDH-catalyzed reaction proceeds rapidly 
to the end point. 
GDH requires a pyridine nucleotide coenzyme such as NAD.sup.+ or 
NADP.sup.+. For example; NAD.sup.+ -dependent GDH (EC 1.1.1.6) from 
Escherichia coli, Klebsiella pneumoniae, Acetobacter suboxydans, or 
Geotrichum candidum [J. Biol, Chem., 203, 153 (1953), ibid., 235, 1820 
(1960), and Agric. Biol, Chem., 46, 3029 (1982)] forms dihydroxyacetone 
from glycerol; NADP.sup.+ -dependent GDH (EC 1.1.1.72) from rabbit 
skeletal muscle [Biochim. Biophys. Acta, 258, 40 (1972)] forms 
D-glyceraldehyde; and NADP.sup.+ -dependent glycerol-2-dehydrogenase (EC 
1.1.1. 156) from a green alga Dunaliella parva [FEBS Letter, 29, 153 
(1973)] forms dihydroxyacetone. While any of the above cited GDHs can be 
used in the invention. GDH from Geotrichum candidum is preferred, due to a 
small Michaelis constant (Km value) for glycerol. 
Suitable phosphate donors for use in the invention include, ATP, 
uridine-5'-triphosphate (UTP), inosine-5'-triphosphate (ITP), 
cytidine-5'-triphosphate (CTP), and guanosine-5'-triphosphate (GTP). ATP 
is best phosphate donor for dihydroxyacetone kinase from 
Schizosaccharomyces pombe IFO 0354. 
For determining glycerol concentration by the above reaction, it is most 
convenient to measure the change in the absorbance at 340 nm due to the 
NAD(P)H formation. Other methods are also applicable such as fluorometry, 
colorimetry by use of phenazine methosulfate and a tetrazolium salt 
[Clinica Chimica Acta, 81, 125 (1977)], and colorimetry by use of 
diaphorase and a tetrazolium salt. 
The composition of the invention for determining glycerol contains an 
enzyme selected from triokinase and dihydroxyacetone kinase, GDH, a 
pyridine nucleotide coenzyme, a phosphate donor, and a divalent metal 
cation, as essential components examples of which are as mentioned above. 
The concentration of each component can be varied over a wide range. For 
instance, suitable concentrations of GDH are 0.01 to 0.5 u/ml when 
glycerol is determined by the rate assay system, and is 0.5 u/ml or more 
when glycerol is determined by the end point assay system. Suitable 
concentrations of triokinase or dihydroxyacetone kinase are 0.1 u/ml and 
more. 
The reaction is carried out at a pH of 7-9 and at a temperature of 
25.degree.-40.degree. C. For the purpose of keeping the pH constant, the 
reaction mixture is desired to contain a buffer. Any buffer may be 
employed for this purpose provided that the pH thereof lies in the 
above-mentioned range, but Tris-HCl buffer (pH 8.0-8.5) is preferable. 
The invention is illustrated in more detail with reference to the following 
examples. Enzymes used in the examples and enzyme assays are described 
below. 
(1) Preparation of dihydroxyacetone kinase 
Schizosaccharomvces pombe IFO 0354 was inoculated to a medium (pH 6.2) 
containing 1% malt extract, 0.3% peptone, 0.1% yeast extract, 0.2% K.sub.2 
HPO.sub.4, 0.05% MgSO.sub.4.7H.sub.2 O, 0.05% KCl, and 0.001% 
FeSO.sub.4.7H.sub.2 O. The cultivation was carried out at 30.degree. C. 
for 48 hours. 
The cells were collected by centrifugation from 10 l of the culture broth, 
suspended in a 20 mM Tris-HCl buffer (pH 7.0), and disrupted with a Dyno 
Mill KDL. The extract was centrifuged to remove the cell debris , then a 
polyethyleneimine solution was added to the supernatant up to a 
concentration of 0.02%, and the resulting precipitate was removed by 
centrifugation. Then ammonium sulfate was added to the supernatant for the 
salting-out of the enzyme, and the enzyme precipitate in the fractions of 
40-70% saturation was harvested. This precipitate was dissolved in the 
same buffer, subjected to a Sephadex G-25 gel filtration for desalting, 
and applied to a DEAE-Sepharose (Pharmacia Fine Chemicals Co.) , column 
equilibrated with the same buffer. After washing the column, the enzyme 
was eluted with a linear gradient of KCl from 0 to 0.3 M. Two peaks of 
enzyme activity were observed with DEAET-Sepharose column chromatography. 
The fractions eluted with 0.12 M KCl (referred to as DHAK(I)) and those 
eluted with the 0.16 M KCl (referred to as DHAK (II)) were independently 
collected. 
DHAK(I) and DHAK(II) were precipitated by adding ammonium sulfate to 
combined fractions up to 80% saturation, respectively. Each enzyme 
precipitates was collected by centrifugation, dissolved in the same 
buffer, and desalted with Sephadex G-25 column chromatography. Then each 
resulting enzyme solution was passed through a Blue-Sepharose (Pharmacia 
Fine Chemical Co.) column previously equilibrated with a 40 mM Tris-HCl 
buffer (pH 7.0), and active fractions were concentrated by 
ultrafiltration. Thus, 120 units of DHAK(I) and 360 units of DHAK(II) were 
obtained. DHAK(I) and DHAK(II) are effective in the mixed state as well. 
In the following examples, the mixture of DHAK(I) and DHAK(II) thereof was 
used. 
Enzyme assay: The standard assay mixture for measuring the activity of DHAK 
consists of 1.0 ml of 0.1 M triethanolamine-HCl buffer (pH 7.5), 2.5 mM 
ATP, 4 mM MgSO.sub.4, 0.2 mM NADH, 1.0 mM DHA, 2.5 units of 
glycerol-3-phosphate dehydrogenase (Boehringer Mannheim GmbH), and 0.01 ml 
of enzyme solution. The reaction is started by adding enzyme solution, and 
the decrease in the absorbance at 340 nm is measured 
spectrophotometrically at 25.degree. C. In the blank assay, DHA is 
excluded from the reaction mixture. One unit of the enzyme activity is 
defined as the amount of enzyme which catalyzes the formation of 1 .mu.mol 
NAD.sup.+ in one minute under the above conditions. The above reaction is 
represented by the following equations: 
##STR2## 
(2) Preparation of triokinase 
Triokinase was prepared from swine liver in accordance with the process 
described in Meth. Enzymol., 5. 362 (1962). The enzyme activity was 
determined in the same manner of dihydroxyacetone kinase except that 
D-glyceraldehyde was used in the place of dihydroxyacetone as a substrate. 
(3) Preparation of glycerol dehydrogenase (GDH) 
GDH from Geotrichum candidum and GDH from rabbit skeletal muscle were 
prepared in accordance with the process described in Agric. Biol. Chem., 
46, 3029 (1982) and the process described in Biochim. Biophys. Acta, 258, 
48 (1972), respectively. One unit of GDH activity is defined as the amount 
of enzyme which catalyzes the formation of 1 .mu.mole of NAD(P)H in one 
minute at 25.degree. C. and pH 8.0 in the reaction of glycerol and 
NAD(P).sup.+ 
(4) Expression of lipase activity 
One unit of lipase activity is defined as the amount of enzyme catalyzing 
the liberation of 1 .mu.mole of the corresponding fatty acid in one minute 
at 37.degree. C. and pH 7.0 when the mixture of olive oil emulsion and 
bovine serum albumin is used as a substrate. 
EXAMPLE 1 
0.02 ml each of glycerol standard solutions in the range from 75 to 1050 
mg/dl as triolein was mixed with 0.96 ml of 0.15 M Tris-HCl buffer (pH 
8.5) containing 1.04 mg/ml MgSO.sub.4.7H.sub.2 O, 2.0 mg/ml NAD and 1.5 
mg/ml ATP, in a 1-ml cuvette. The mixture was kept at 37.degree. C. for 3 
minutes, then 0.02 ml of 0.02 M Tris-HCl buffer (pH 7.5) containing 8.75 
u/ml GDH of Geotricum candidum and 25 u /ml dihydroxyacetone kinase was 
added to the reaction mixture, and the increase in the absorbance at 340 
nm was measured. For comparison, the above procedure was repeated on the 
same reaction system without dihydroxyacetone kinase. 
Relations between the reaction time and the absorbance are shown in FIGS. 1 
(example of the invention) and 2 (comparative example)respectively, and 
the calibration curve according to the method of the invention is shown in 
FIG. 3. 
It shows that, according to the method of the invention, the reaction rate 
is constant with respect to the reaction time even at a glycerol 
concentration as triolein) of higher than 1000 mg/dl and glycerol can be 
quantitatively determined with a high sensitivity; while according to the 
method of the comparative example, the reaction rate is low and decreases 
with time. 
EXAMPLE 2 
Glycerol was determined in the same manner as in Example 1 using 
triokinase, GDH of rabbit skeletal muscle, and NADP.sup.+ in the place of 
dihydroxyacetone kinase, GDH of Geotrichum candidum, and NAD, 
respectively. The results were similar to those of Example 1. 
EXAMPLE 3 
Glycerol was determined in the same manner as in Example 1 using 
CaCl.sub.2, CoCl.sub.2, and MnCl.sub.2 separately in the place of 
MgSO.sub.4. In all the cases, the results were similar to those of Example 
1. 
EXAMPLE 4 
In test tubes, 1.96 ml portions of a 0.15 M Tris-HCl buffer (pH 8.5) 
containing 1.04 mg/ml MgSO.sub.4. 7H.sub.2 O, 2.0 mg/ml NAD, and 1.5 mg/ml 
ATP were mixed with 0.02 ml portions of 0.02 M Tris-HCl buffer (pH 7.5) 
containing 75 u/ml GDH of Geotrichum candidum and 25 u/ml dihydroxyacetone 
kinase. Each mixture was maintained at 37.degree. C. for 3 minutes, then 
0.02 ml each of standard glycerol solutions in the range from 75 to 900 
mg/dl (as triolein) was added, the reaction was allowed to proceed for 20 
minutes at 37.degree. C. and the increase in the absorbance at 340 nm 
after 20 minutes was measured. For comparison, the above procedure was 
repeated on the same reaction system without dihydroxyacetone kinase. 
As shown in FIG. 4, the results shows that; according to the method of the 
invention, the GDH-catalyzed reaction proceeds to the end point, the 
calibration curve is hence linear up to a glycerol concentration of at 
least 900 mg/dl (as triolein), and a high sensitive determination of 
glycerol is possible; while the method of the comparative example results 
in low reactivity, poor sensitivity, and a non-linear calibration curve. 
EXAMPLE 5 
Determination of triglyceride 
Triglyceride concentration was determined by adding lipase (lipoprotein 
lipase) to samples to hydrolyze the triglyceride, and measuring the 
quantities of formed glycerol, as follows: 
0.02 ml each of serum samples was mixed with 0.96 ml of 0.15 M Tris-HCl 
buffer (pH 8.5) containing 500 u/ml lipoprotein lipase (Amano 
Pharmaceutical Co., Ltd.), 1.04 mg/ml MgSO.sub.4.7H.sub.2 O, 2.0 mg/ml 
NAD, 1.5 mg/ml ATP, and 0.1% Triton X-100, and the mixture was incubated 
at 37.degree. C. for 5 minutes. Then, 0.02 ml of 0.02 M Tris-HCl buffer 
(pH 7.5) containing 8.75 u/ml GDH of Geotrichum candidum and 25 u/ml 
dihydroxyacetone kinase was added to the mixture, and the increase in the 
absorbance at 340 nm was measured. In addition, the calibration curve 
shown in FIG. 5 was made by repeating the above procedure using standard 
serum samples. Triglyceride contents in the above serum samples were 
determined from the calibration curve. 
Further, comparison studies were carried out with serum samples between the 
method of the invention and the other known method [TG Kit-K (tradename), 
containing lipoprotein lipase, glycerol kinase, and glycerol-3-phosphate 
oxidase, supplied by Nihon Shoji Co., Ltd.]. As shown in FIG. 6, the 
correlation between the two-methods was excellent.