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An enzymatic, spectrophotometric glycerol assay with increased basic sensitivity
.\NALYTlCAL
BIOCHEMISTRY
An
Enzymatic,
59,
248-258
Spectrophotometric
with
Increased
F. MOLLER Department
of
Hcccived
(1974)
Biochemistry,
June
26, 1973;
Glycerol
Basic AKD
hf.
Queen’s
accepted
Assay
Sensitivity ROOM1
w. Universily,
Kovembcr
Kingston,
Ontario
28, 1973
Glycerol can be assayed with good precision and accuracy in biological samples free of dihydroxgacetone using a series of four enzymatic reactions. Two moles of nicotinamide-adenine dinucleotide are reduced per mole of glycerol, thus doubling the basic sensitivity of existing enzymatic. spectrophotomctric methods. The method was developed primarily for use in studies of adipose tissue. No deproteinization was required in samples obtained from incubation of isolated fat cells or pieces of adipose tissue.
Several sensitive and specific enzymatic methods have been developed for the assay of glycerol in biological materials (l-4). Kicotinamide-adenine dinucleotide (NAD) may be reduced by glycerol in two steps as in Wieland’s method (1) involving glycerokinase (GK) and glycerol-3phosphate dehydrogenase (GDH) (Reactions 1 and 2 below), or in one step catalyzed by glycerol dehydrogenase as in the method of HimmsHagen and Hagen (2). In these t#wo methods the reduction of the dinuclcotidc is measured spectrophotomet,rically but it may also with greater sensitivity bc measured fluorimetrically as in Laurel1 and Tibbling’s modification (5) of Wieland’s method. In a third method (3,4) adenosine 5’-diphosphate (ADP) formed in the enzymatic phosphorylation of glycerol reacts in a second step with phosphoenolpyrurate in the pyruvate kinase reaction and the resulting pyruvate finally oxidizes NADH in the third step cat,alyzed by lactate dehydrogenase. In contrast to the first two methods the forward reaction of each step in the last method is favored thermodynamically resulting in the rapid stoichiometric oxidation of XADH. Although dihydroxyacetone would be expected to react in the third method its phosphorylation appears to be too slow to cause interference (4). Deproteinization of samples when required is usually done in several steps with either perchloric acid (l-3)) or trichloroacetic acid (4) or zinc sulphat,e and barium hydroxide (5). For deproteinization in a single st’ep metaphosphoric acid 16,7), heat denaturation (7)) or ultrafiltration (7) may he used. Copyright All rights
@ 1974 by Academic Press, of wproduction in any form
248 Inc. reserved.
EKZ
1‘1RIATIC
ASSAY
FOR
249
GLYCEROL
The purpose of this report is to describe an enzymatic method in which the yield of NADH is doubled as compared with presently used enzymatic methods; this decreases the basic limit. of detection of glycerol. An added advantage of the procedure is that deproteinization is possible in a single step with tungstic acid. The method was worked out with a view to its application in studies of adipose tissue where only small samples may be available for analysis such as in perfusion studies or in work with isolated fat cells or pieces of adipose tissue. The method is based on the following series of reactions: G Ii
Glycerol r,-wglycerophosphate
+ ATP + NAD
-
r,ol-glycerophosphate GDH
-
+ i\I)P
dihydrouyacetoncphosphate
(1) + NADH (2)
TIM
dihydroxyacetonephosphate
-
n-glyceraldehyde-s-phosphate
(3)
HAbO4
wglyceraldehyde-s-phosphate
+ NAD
GPDH
-
glyrerate-s-phosphate
+ NAI>H
(4)
At high NAD concentrations and in the presence of arsenate Reaction 4 proceeds essentially to completion (8). In principle the method is a further derelopmcnt of Wieland’s met8hod (1). X4TERL4LS
AND
RIETHODS
Stock Reagents’ Tria [tris (hydroxymethyl) aminomethane] 1 N ; MgCl, 0.021 M ; sodium arsenate (?;a,HAsO,l) 0.04 M; glycerol 4 InAt (prepared by weighing) ; GDH and TIM (triosephosphate isomerase) in mixed suspension in 2.4 M ammonium sulphase (2 mg protein/ml, 165 U GDH/mg protein, 107 U TIMimg protein; GPDH (glyceraldehyde phosphate, dehydrogenase) in 2.5 M INH,),SO, (10 mg proteiw’ml, 90 V/mg protein) ; GK in 2.4 M (NH,),SO, (5 mg protein/ml, 85 U/mg protein) ; tungstic acid, commercially prepared reagent stabilized either with sulphuric acid’ or phosphoric acid.” The reagent) is diluted according to the sample prot’ein concentration as recommended for blood or serum by the manufacturer. The following solutions were prepared shortly before use: Tris 0.2 31; mcrcaptoethanol 0.04 M. Reagent mixture: 30 mg ATP (disodium salt) and 60 mg NAD dissolved in a mixture of 7.5 ml Tris (0.2 M) , 2 ml ?\lgCL and 2 ml sodium arsenak. The pH of this solution is adjusted to 9.2 with ‘Enzymes and coenzymes were purchased Louis. MO. Other reagents used were of highest ’ Hycel. Inc., Houston, TX. ’ Canlab, Toronto, Ontario.
from Sigma Chemical purity available.
Company,
St.
250
MOLLER
AXD
ROOM1
1 N KOH and the volume made to 14 ml with water. Enzyme mixture: 15 /ill of the mixed suspension of GDH and TIM is added to 0.2 ml icecold mercaptoethanol solution. To this mixture is further added 15 ,J of the GPDH suspension. Glycerol kinase: 30 ,~l GK suspension is added to 0.2 ml ice-cold mercaptoethanol solution. Glycerol working standards are prepared by dilution of the stock standard. Procedure When protein precipitation is required 1 vol of sample is mixed with 9 vol of tungstic acid reagent. The sample is left for 10 min and then centrifuged. The assay is performed in semi-micro glass cuvettes adding reagents, etc. as follows: Sample :
Reagent Water: Enzyme
mixture: mixture :
0.1 ml or less (with protein) or 0.4 ml or less (deproteinized) 0.7 ml to make volume 1.1 ml 0.02 ml
The optical density is then measured at 340 nm and after two identical readings 15-20 min apart 0.02 ml glycerol kinase is added and the optical density read after 1, 2, and possibly 3 hr. There is usually no reaction due to the addition of enzyme mixture and the glycerol kinase can be added after two initial readings. Two glycerol standards are assayed in duplicate with each set of samples. The volume of protein containing solution used above (0.1 ml) is suitable for a 4% albumin solution. A satisfact’ory assay can also be performed using 0.2 ml 4% albumin solution and correcting for the small difference in volume. However, there is a slight increase in the initial optical density under these circumstances. The concentration of glycerol in the sample is calculated after completion of the reaction from t’he change in optical density after adding glycerol kinase and from the molecular extinction coefficient for NADH (6.22 X 106cm2/mole) (9). RESULTS
ilND
DISCUSSION
Progress curves obtained with various amounts of glycerol in water are shown in Fig. 1. These curves indicate that the reaction was complete within 2 hr when less than about 1.2 X lo-” pmole of glycerol was assayed; this corresponds to concentrations in deproteinized and non-deproteinized samples of 0.3 and 0.12 mM respectively. Completion required 3 hr when the concentrations were higher. A standard curve prepared from changes in optical densities obtained after 3 hr is shown in Fig. 2.
ENZYMATIC
ASSAT
FOR
GLYCEROL
FIG. 1. Time course of reduction of NAD by glycerol after xddition kinase. Conditions as described under procedure.
251
of glycerol
The standard curve was linear with the range of glycerol amounts assayed (0.002-0.02 pmole). The mean recovery on the basis of the extinction coefficient given above was 93.5 + 1.38% (SEM) in this experiment.
Accuracy Since the method was intended for use primarily in studies of adipose tissue including the analysis of perfusates and since these would probably require protein precipitation, as would serum or plasma, the accuracy of the procedure involving protein precipitation was studied. A freeze-dried human control serum” was reconstituted with water or with a glycerol solution and the recovery of added glycerol was determined. When the control glycerol concentration was increased by about 50% (n = 8) the mean recovery was 94.4% and the standard error of the mean was &2.96% (Table 1). The recovery was thus essentially the same from aqueous so-
Glycerol
~~rnoles
x 10e3
FIG. 2. Relationship between amount of glycerol in cuvette and change in optical density 3 hr after adding glycerol kinase. Conditions as described under procedure. ’ BDH Chemicals, Toronto,
Ontario.
252
MOLLER
Recovery
Number of assays
Sample Serum
control
Serum + 0.16 glycerol/ml
2 pmoles
6
AND
TABLE of Glycerol
ROOM1
1 Added
to Rerrlmf’
Mean glycerol cone &moles/ml) o’267 0.418
i 0.272 0.262 k 0.0045b
Mean recovery Cfimoles, ‘ml)
0.151
a A freeze-dried clinical control serum was reconstitut,ed to give the concentrations indicated in the first c~olnmn b Standard error of the mean.
f 0.0045b
with water of the table.
Mean recovery (7; of added glycerol)
94.4
+ 2.96*
or glycerol
solution
lutions (standard curve) and from biological samples indicating that recovery from biological samples calculated from a standard curve would be close to 100%. Since any dihydroxyacetone present in the serum would increase the apparent glycerol concentration (see below), t,he control serum used above was analyzed for dihpdroxyacetone by a slight modification of the method of Wieland (10). The optical density readings obtained were too low, however, to be of significance indicating at the most a trace of dihydroxyacet’one. In this analysis tungstic acid instead of the prescribed perchloric acid (10) was used for protein precipitation. Dihydroxyacetone was fully recovered in such deproteinized samples. Precision We found that the Krebs-Ringer buffer (bicarbonate or phosphate) with 4% bovine albumin, used in this laboratory for incubation of isolated fat cells or pieces of adipose tissue, did not’ require deproteinization before assay for glycerol. The precision of this procedure was determined by duplicate analysis of 10 incubation media. The results given in Table 2 indicate good reproducibility. As with serum it was important to establish whether or not dihydroxyacetone was present in such samples. Using the procedure described by Wieland (10) dihydroxyacetone was virtually undetectable in the media used to incubate adipose tissue. The data from one experiment using pieces of adipose tissue pooled from the epididymal fat pads of four fed rats are given in Table 3. Although the values for dihydroxyacetone would indicate that this might amount to 7% of the measured glycerol release from unstimulated adipose tissue this percentage is of doubtful significance since it was derived from an optical density change of less than 0.01. It should also be noted t’hat the amount of glycerol released from unstimulated adipose tissue was very low as would be expected for fed rats. mhen adipose tissue was stimulated
ENZYMATIC
Precision Number of samples
of Glycerol
Xumber assays
of (n)
20
10
Assay
ASSAY
FOR
TABLF: Using
2 Non-l)eproteirlixed
Cone range (fimoles,/ml 0.047Y~.
j
253
GLYCEROL
Samples‘
-
Mean cnnc (pmules/ml)
Precision* &moles//ml)
0. 13s
f0.0017
212
a Analysis of media used for incubation of pieres of IIon-kmulated and epinephrine The medilun was stimulat,ed adipose tissue (70-80 mg tinslle in 2.25 ml medi~rm). Krebs-Ringer phosphat,e buffer wit,h 4r; bovine albrunill. * Precision was taken ti (~d,“,‘n) I’S where r/ is the difference heiween duplicates and n is the number of assays (15).
by epinephrine (Table 3) relca~e of clihydroxyacetone was similarly questionable while there was an almost tenfold increase in glycerol release. The finding of, at most, trace amounts of dihydroxyacetone in serum and in media used for incubation of nclil~osetissue is consistent with recent reports indicating that dihydroxyacetone levels are very low in tissues (11,12). Sensitivity
Using the mean of 0.418 and the standard error of the mean of 0.0045 (Table 1) the 95% confidence limits using deproteinized samples would be + 0.0106 pnole/ml. This would indicate that glycerol concentrations below about 0.011 pmoleJm1 would be indistinguishable from zero. This sensitivity is about half of that of Laurel1 and Tibbling’s fluorimetric method. Since the precision was better when removal of protein was not
Release
TABLE and I)ihydlox~-nretolle
of Glycerol
Sample Unstimulated adipose tissue Epinephrine st,imulated adipose
Number of samples
Mean glycerol release (pmoles,/g1
3 from
Adipose
Tkrue
llIcan dihydrosyacetone release (as gl!-cerol) (pmoler !g) 3 0.022
2 4
11.70
+ 0.286h
0. 0116 0.170 0.119 + O.WIY
III Vitroa
1 )ihydroxyaretolle release (t-l of glycerol release) 7 :: 11
a Pieces of adipose tissue (70-80 mg) pooled from the epididymal fat pads of four fed rats were incubated in Krebs-Ringer phosphate buffer with4:; bovine albumin (2.25 ml). After incubation for 1 hr the media were assayed for glycerol and dihydrosyacetone. * SEM. c Epinephrine cone was 1 pg/ml.
254
MOLLER
AND
ROOM1
required and since five times more sample could be used the lower for such samples would not be more than 0.002 ~moleJm1. Specificity
limits
and Interference
Table 4 lists compounds which were tested for interference with the assay of glycerol. Ethanol reduced NAD but to an insignificant degree as compared with glycerol. Dihydroxyacetone which is also phosphorylated by glycerol kinase (13) reduced NAD as expected. However, the yield was only about 60% of the theoretical (1 mole NAD reduced per mole dihydroxyacetone). The incomplete reaction with dihydroxyacetone is presumably due to a lower rate of phosphorylation (4). At the concentrations indicated in Table 4 dihydroxyacetone also slightly inhibited (less than 10%) the reduction of NAD by glycerol. PH Optimum reaction rat’es were achieved when the pH of the reagent mixture was adjusted t,o 9.2 as described under reagents. The final pH in the assay mixture under these circumstances was 8.65-8.70. A slightly higher pH (8.85) did not result in significant increases in reaction rate or final yield of NADH. At lower final pH values the yield of NADH decreased as indicated in Fig. 3. Effect of SW Groups The presence of an SH-compound dithiothreitol and mercaptoethanol
Various
Compounds
Final Compound Glucose Fructose n-glyceraldehyde Dihydroxyacetone Ethyleneglycol Ethanol Lactate P-Hydroxybutyrate Acetone a The decrease
Test,ed
is an absolute requirement. Cysteine, are all effective. Cysteine, which was
TABLE 4 for Int,erference
with
t,he Assay
of Glycerol
bM)
Reduction of NADH equiv t’o glycerol cone (mM)
Inhibition (at glycerol cone of 0.01 rnM)
1.69 1.69 0.02 0.02 0.60 16.90 3.38 3.38 3.38
0 0 0 0.006 0 0.001 0 0 0
0 0 0 slight* 0 0 0 0 0
cone in cuvette
degree of inhibition was not assessed of the combined recovery of glycerol
but’ was less than 10% and dihydroxyacetone
as indicated in a mixed
by the sample.
ENZYMATIC
ASSAT
FOR
!-A 75
8-O
255
GLYCEROL
8.5
9.0
PH
F’N. 3. lnfluencc of pH on reduction of NAD by plywrol. Glycerol was assayed as desrribrd under procedure esccpt that cystcine at a final concentration of 6.9 mM (triangles) was used in some espcrimcnts instead of mercaptoethanol (spheres). The pH values refer to the final pH in tllc, complete assay mixture.
used originally in these studies at, a final concentration of 6.9 mrq occasionally caused formation of a precipitate (probably of cystine) and at lower concentrations high blank reactions were obtained. This blank reaction was presumably due to the formation of a complex between NAD and cysteine (14). The blank reaction observed with mercaptoethanol, which can be used at a much lower concentration than cysteine, is probably due to a similar complex formation. Since the rate of complex formation increases rapidly between pH 8 and 10 (11) this reaction may place an upper limit on the pH in the assay. At the presently used mercaptoethanol concentration t’he blank reaction is constant and equivalent to about 2.5 X lo-” pmole glycerol in the assay. The catalytic system with albumin in samples was fully active for at least 2 hr since the appearance rates of NADH and recoveries of glycerol did not differ when identical samples of glycerol were added at rb~ hr intervals during this period to preincubated complete assay mixtures (results not shown). However, some batches of the mixed GDH and TIM preparation appeared to be labile \vhcn albumin was absent from samples.
The present method was compared with Wieland’s method (16) in four experiments. In one experiment aqueous solutions of glycerol were analyzed in triplicate by bot8h methods (Table 5). The regression lines were determined using the four points obtained by each analysis and estimated glycerol concentrations were derived from the regression line. The standard errors of estimate determined for each set of data indicate
256
Comparison
MOLLER
with
Wieland’s
Method
Wieland’s
AND
ROOM1
TABLE 5 and Analysis
of Glycerol
metshod
Standards
Present,
in Water
method
_. Glycerol cone (PM)
Optical density
Estimat,ed” couc (FM)
% of sample cone
Optical density
Estimat.ed” cone (GM)
:;, of sample cone
10 20 40 SO
0.014 0.029 0.061 0.121
9.3 19.3 40.7 so.7
93.0 96.5 101,s 100.9
0.042 0.083 0.161 0.320
10.4 20.5 39.9 79.2
104.0 102.5 99.x 99.0
Mean SEE” a Estimated b Standard
recovery: = +0.70
94.77,
from regression error of estimate.
Mean SEE”
recovery: = to.52
93.3yc
line.
that the present method in this experiment was somewhat more precise than Wieland’s method. Furt.her comparisons with Wieland’s method were made (Table 6) using samples obt,ained from incubations of isolated fat cells stimulated with epinephrine in Krebs-Ringer phosphate buffer. In the first experiment in Table 6, Wieland’s method was followed exactly as described in Ref. 16. In this experiment the mean glycerol concentration obtained
Comparison
with
TABLE 6 Wieland’s Met,hod and Analysis in Media Conkining Albumin Mean glycerol concentration b.Wml) ___-
+ SE&I (yO of mean)
Standards mean recovery (o/l)
s s
0.105 0.092
1.14 0.77
93.8 92.6
5 5
0.163 0.160
0.51 1.00
90.0 95.5
r
0.194 0.194
0.5s 0.97
97.7 95.1
Replicate samples Expt la Wieland’s method Present method Expt 2 Wieland’s method Present method Expt 3 Wieland’s method Present method
of (ilycerol
.G
a Samples analyzed were Krebs-Ringer phosphate albumin (4%) media separated from isolated rat, fat cells after one hour of incubat,ion with ephinephrine. The samples in all three experiments were analyzed by the present method wkhout deproteinizatinn. The samples were deprot,einized with perchloric acid in Expt 1, by ultrafiltration in Expt 2, and t.hey were not deproteinized in Expt 3 using Wieland’s method.
EXZYMATIC
ASSAY
FOR
GLYCEROL
257
by the present method was 87.6% of that obtained by Wieland’s method. However, the recoveries of glycerol in standards (made with KrcbsRinger phosphate buffer cont,aining 4% albumin) were nearly identical by the two methods. We thought on this basis that the higher values ohtained by Wieland’s method might be due to hydrolysis of glycerides during the precipitation of proteins with pcrchloric acid. To see whether this was the case samples from a second experiment and standards Fvere deproteinized by ultrafiltration through an Amicon PIZI-10 filter and analyzed by Wieland’s method. The results (Espt 2, Table 6) were nearly identical with the resulk obtained by the present method using non-deprot,einized samples. It nppcared, however, from t,hc recovery of glycerol in standards that there was a slight loss (appros 50/c of glycero1 in ultrafiltered samples. Since such a 10s~ ‘q might be concentration dependent the small difference between the results of the two methods could be due to variable degrees of losses in samples and rtandards. In the third experiment enzymatic analyses were therefore conducted direct,ly on non-clcprotcinizcd samples by both methods and identical results were obtained (Expt 3, Table 6). It would t8hus appear that Wieland’s method using dcproteinization with perchloric arid would tend to give too high values at least in samples obtained as described here. The two methods agrectl well on the other hand when precautions wcrc taken to minimize breakdown of glyccritlrs. In this comparison the present met,hod was more precise (and accurate‘) than the standard Wielnnd method (16) involving protein precipitation with perchloric acid. It would he expected that, the method in general would be more precise than methods yielding only 1 mole of NASH per mole of glycerol since the resuks by all methods for glyecrol rlcpcnd on differences betiveen initial optical densities before addit’ion of the enzyme specific for glycerol and the optican densities at t,he completion of t,he rcact’ion. This would bc particularly true of course for samples with low glycerol concentrations.
A new method for the analysis of glycerol is described. Employing a series of four enzymatic reactions its basic sensitivity is twice that. of existing enzymatic methods. In addition protein precipitation, when necessary, is possible in one step with tungstic acid. Dihydrosyacetone, which reacts incompletely in the assay, would have to he absent, from samples. This requirement was satisfied in samples ohtained from incubation of adipose tissue and in a clinical control serum. Although the reaction time is relatively lon,(r the method should he a useful epectrophotometric alternative to fluorimctrir methods where very low glycerol
258
MOLLER
AND
ROOM1
levels are encountered as, for example, in studies of basal (unstimulated) lipolysis when only small amounts of adipose tissue or small numbers of isolated fat cells are available. It would appear useful as well in analysis of tissue extracts for glycerol although the deproteinization required for such samples, because of the presence of various dehydrogenases and their substrates, would make it less sensitive than fluorimetric methods. ACKNOWLEDGMENT Financial support acknowledged.
from the Medical
Research Council
of Canada is gratefully
REFERENCES 1. WIELAND, 2. 3. 4.
5. 6. 7. 8.
0. (1957) Biochem. Z. 329, 313. HIMMS-HAGEN, J., AND HAGEN, P. B. (1962) Can. J. Biochem. Physiol. 40, 1129. KREUTZ, F. H. (1962) Klin. Wochenschr. 40, 362. GARLAND, P. B., AND RANDLE, P. J. (1962) Nature (London) 196, 987. LAURELL, S., AND TIBBLING, G. (1966) Clin. Chim. Acta 13, 317. SPINELLA, C. J., AND MAGER, M. (1966) J. Lipid Res. 7, 167. FARESE, G., AND MAGER, M. (1970) J. Lipid Res. 11, 274. VELICX, S. F. (1955) in Methods in Enzymology (Colowick, S. P., and Kaplan,
N. O., eds.), Vol. 1, p. 401. Academic Press, New York. 9. HORECKER, 13. L.. AND HORNBERG, A. (1948) J. Biol. Chem. 175, 385. 10. WIELAND, 0. (1965) irL Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), p. 244, Academic Press, New York. 11. WALTON, D. J.. AND GAUCHIE. L. M. (1972) Anal. Biochem. 46, 352. 12. CHARLTON, J. M., AND VAN H~YNINGEX, R. (1969) Anal. Biochem. 30, 313. 13. BERGMEYER, H. U., HOLZ, G.. KAUDER. E. M.. M~LLERING, H.. AND WIELAND, 0. (1961) Biochem. Z. 333, 471. 14. V.~N EYS, J., AND KAPLAN, N. 0. (1957) J. Biol. Chem. 228, 305. 15. HENRY, R. J. (1964) Clinical Chemistry. Principles and Technics, p. 125, Harper and Row, New York. 16. WIELAND, 0. (1965) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), p. 211, Academic Press, New York.
BIOCHEMISTRY
An
Enzymatic,
59,
248-258
Spectrophotometric
with
Increased
F. MOLLER Department
of
Hcccived
(1974)
Biochemistry,
June
26, 1973;
Glycerol
Basic AKD
hf.
Queen’s
accepted
Assay
Sensitivity ROOM1
w. Universily,
Kovembcr
Kingston,
Ontario
28, 1973
Glycerol can be assayed with good precision and accuracy in biological samples free of dihydroxgacetone using a series of four enzymatic reactions. Two moles of nicotinamide-adenine dinucleotide are reduced per mole of glycerol, thus doubling the basic sensitivity of existing enzymatic. spectrophotomctric methods. The method was developed primarily for use in studies of adipose tissue. No deproteinization was required in samples obtained from incubation of isolated fat cells or pieces of adipose tissue.
Several sensitive and specific enzymatic methods have been developed for the assay of glycerol in biological materials (l-4). Kicotinamide-adenine dinucleotide (NAD) may be reduced by glycerol in two steps as in Wieland’s method (1) involving glycerokinase (GK) and glycerol-3phosphate dehydrogenase (GDH) (Reactions 1 and 2 below), or in one step catalyzed by glycerol dehydrogenase as in the method of HimmsHagen and Hagen (2). In these t#wo methods the reduction of the dinuclcotidc is measured spectrophotomet,rically but it may also with greater sensitivity bc measured fluorimetrically as in Laurel1 and Tibbling’s modification (5) of Wieland’s method. In a third method (3,4) adenosine 5’-diphosphate (ADP) formed in the enzymatic phosphorylation of glycerol reacts in a second step with phosphoenolpyrurate in the pyruvate kinase reaction and the resulting pyruvate finally oxidizes NADH in the third step cat,alyzed by lactate dehydrogenase. In contrast to the first two methods the forward reaction of each step in the last method is favored thermodynamically resulting in the rapid stoichiometric oxidation of XADH. Although dihydroxyacetone would be expected to react in the third method its phosphorylation appears to be too slow to cause interference (4). Deproteinization of samples when required is usually done in several steps with either perchloric acid (l-3)) or trichloroacetic acid (4) or zinc sulphat,e and barium hydroxide (5). For deproteinization in a single st’ep metaphosphoric acid 16,7), heat denaturation (7)) or ultrafiltration (7) may he used. Copyright All rights
@ 1974 by Academic Press, of wproduction in any form
248 Inc. reserved.
EKZ
1‘1RIATIC
ASSAY
FOR
249
GLYCEROL
The purpose of this report is to describe an enzymatic method in which the yield of NADH is doubled as compared with presently used enzymatic methods; this decreases the basic limit. of detection of glycerol. An added advantage of the procedure is that deproteinization is possible in a single step with tungstic acid. The method was worked out with a view to its application in studies of adipose tissue where only small samples may be available for analysis such as in perfusion studies or in work with isolated fat cells or pieces of adipose tissue. The method is based on the following series of reactions: G Ii
Glycerol r,-wglycerophosphate
+ ATP + NAD
-
r,ol-glycerophosphate GDH
-
+ i\I)P
dihydrouyacetoncphosphate
(1) + NADH (2)
TIM
dihydroxyacetonephosphate
-
n-glyceraldehyde-s-phosphate
(3)
HAbO4
wglyceraldehyde-s-phosphate
+ NAD
GPDH
-
glyrerate-s-phosphate
+ NAI>H
(4)
At high NAD concentrations and in the presence of arsenate Reaction 4 proceeds essentially to completion (8). In principle the method is a further derelopmcnt of Wieland’s met8hod (1). X4TERL4LS
AND
RIETHODS
Stock Reagents’ Tria [tris (hydroxymethyl) aminomethane] 1 N ; MgCl, 0.021 M ; sodium arsenate (?;a,HAsO,l) 0.04 M; glycerol 4 InAt (prepared by weighing) ; GDH and TIM (triosephosphate isomerase) in mixed suspension in 2.4 M ammonium sulphase (2 mg protein/ml, 165 U GDH/mg protein, 107 U TIMimg protein; GPDH (glyceraldehyde phosphate, dehydrogenase) in 2.5 M INH,),SO, (10 mg proteiw’ml, 90 V/mg protein) ; GK in 2.4 M (NH,),SO, (5 mg protein/ml, 85 U/mg protein) ; tungstic acid, commercially prepared reagent stabilized either with sulphuric acid’ or phosphoric acid.” The reagent) is diluted according to the sample prot’ein concentration as recommended for blood or serum by the manufacturer. The following solutions were prepared shortly before use: Tris 0.2 31; mcrcaptoethanol 0.04 M. Reagent mixture: 30 mg ATP (disodium salt) and 60 mg NAD dissolved in a mixture of 7.5 ml Tris (0.2 M) , 2 ml ?\lgCL and 2 ml sodium arsenak. The pH of this solution is adjusted to 9.2 with ‘Enzymes and coenzymes were purchased Louis. MO. Other reagents used were of highest ’ Hycel. Inc., Houston, TX. ’ Canlab, Toronto, Ontario.
from Sigma Chemical purity available.
Company,
St.
250
MOLLER
AXD
ROOM1
1 N KOH and the volume made to 14 ml with water. Enzyme mixture: 15 /ill of the mixed suspension of GDH and TIM is added to 0.2 ml icecold mercaptoethanol solution. To this mixture is further added 15 ,J of the GPDH suspension. Glycerol kinase: 30 ,~l GK suspension is added to 0.2 ml ice-cold mercaptoethanol solution. Glycerol working standards are prepared by dilution of the stock standard. Procedure When protein precipitation is required 1 vol of sample is mixed with 9 vol of tungstic acid reagent. The sample is left for 10 min and then centrifuged. The assay is performed in semi-micro glass cuvettes adding reagents, etc. as follows: Sample :
Reagent Water: Enzyme
mixture: mixture :
0.1 ml or less (with protein) or 0.4 ml or less (deproteinized) 0.7 ml to make volume 1.1 ml 0.02 ml
The optical density is then measured at 340 nm and after two identical readings 15-20 min apart 0.02 ml glycerol kinase is added and the optical density read after 1, 2, and possibly 3 hr. There is usually no reaction due to the addition of enzyme mixture and the glycerol kinase can be added after two initial readings. Two glycerol standards are assayed in duplicate with each set of samples. The volume of protein containing solution used above (0.1 ml) is suitable for a 4% albumin solution. A satisfact’ory assay can also be performed using 0.2 ml 4% albumin solution and correcting for the small difference in volume. However, there is a slight increase in the initial optical density under these circumstances. The concentration of glycerol in the sample is calculated after completion of the reaction from t’he change in optical density after adding glycerol kinase and from the molecular extinction coefficient for NADH (6.22 X 106cm2/mole) (9). RESULTS
ilND
DISCUSSION
Progress curves obtained with various amounts of glycerol in water are shown in Fig. 1. These curves indicate that the reaction was complete within 2 hr when less than about 1.2 X lo-” pmole of glycerol was assayed; this corresponds to concentrations in deproteinized and non-deproteinized samples of 0.3 and 0.12 mM respectively. Completion required 3 hr when the concentrations were higher. A standard curve prepared from changes in optical densities obtained after 3 hr is shown in Fig. 2.
ENZYMATIC
ASSAT
FOR
GLYCEROL
FIG. 1. Time course of reduction of NAD by glycerol after xddition kinase. Conditions as described under procedure.
251
of glycerol
The standard curve was linear with the range of glycerol amounts assayed (0.002-0.02 pmole). The mean recovery on the basis of the extinction coefficient given above was 93.5 + 1.38% (SEM) in this experiment.
Accuracy Since the method was intended for use primarily in studies of adipose tissue including the analysis of perfusates and since these would probably require protein precipitation, as would serum or plasma, the accuracy of the procedure involving protein precipitation was studied. A freeze-dried human control serum” was reconstituted with water or with a glycerol solution and the recovery of added glycerol was determined. When the control glycerol concentration was increased by about 50% (n = 8) the mean recovery was 94.4% and the standard error of the mean was &2.96% (Table 1). The recovery was thus essentially the same from aqueous so-
Glycerol
~~rnoles
x 10e3
FIG. 2. Relationship between amount of glycerol in cuvette and change in optical density 3 hr after adding glycerol kinase. Conditions as described under procedure. ’ BDH Chemicals, Toronto,
Ontario.
252
MOLLER
Recovery
Number of assays
Sample Serum
control
Serum + 0.16 glycerol/ml
2 pmoles
6
AND
TABLE of Glycerol
ROOM1
1 Added
to Rerrlmf’
Mean glycerol cone &moles/ml) o’267 0.418
i 0.272 0.262 k 0.0045b
Mean recovery Cfimoles, ‘ml)
0.151
a A freeze-dried clinical control serum was reconstitut,ed to give the concentrations indicated in the first c~olnmn b Standard error of the mean.
f 0.0045b
with water of the table.
Mean recovery (7; of added glycerol)
94.4
+ 2.96*
or glycerol
solution
lutions (standard curve) and from biological samples indicating that recovery from biological samples calculated from a standard curve would be close to 100%. Since any dihydroxyacetone present in the serum would increase the apparent glycerol concentration (see below), t,he control serum used above was analyzed for dihpdroxyacetone by a slight modification of the method of Wieland (10). The optical density readings obtained were too low, however, to be of significance indicating at the most a trace of dihydroxyacet’one. In this analysis tungstic acid instead of the prescribed perchloric acid (10) was used for protein precipitation. Dihydroxyacetone was fully recovered in such deproteinized samples. Precision We found that the Krebs-Ringer buffer (bicarbonate or phosphate) with 4% bovine albumin, used in this laboratory for incubation of isolated fat cells or pieces of adipose tissue, did not’ require deproteinization before assay for glycerol. The precision of this procedure was determined by duplicate analysis of 10 incubation media. The results given in Table 2 indicate good reproducibility. As with serum it was important to establish whether or not dihydroxyacetone was present in such samples. Using the procedure described by Wieland (10) dihydroxyacetone was virtually undetectable in the media used to incubate adipose tissue. The data from one experiment using pieces of adipose tissue pooled from the epididymal fat pads of four fed rats are given in Table 3. Although the values for dihydroxyacetone would indicate that this might amount to 7% of the measured glycerol release from unstimulated adipose tissue this percentage is of doubtful significance since it was derived from an optical density change of less than 0.01. It should also be noted t’hat the amount of glycerol released from unstimulated adipose tissue was very low as would be expected for fed rats. mhen adipose tissue was stimulated
ENZYMATIC
Precision Number of samples
of Glycerol
Xumber assays
of (n)
20
10
Assay
ASSAY
FOR
TABLF: Using
2 Non-l)eproteirlixed
Cone range (fimoles,/ml 0.047Y~.
j
253
GLYCEROL
Samples‘
-
Mean cnnc (pmules/ml)
Precision* &moles//ml)
0. 13s
f0.0017
212
a Analysis of media used for incubation of pieres of IIon-kmulated and epinephrine The medilun was stimulat,ed adipose tissue (70-80 mg tinslle in 2.25 ml medi~rm). Krebs-Ringer phosphat,e buffer wit,h 4r; bovine albrunill. * Precision was taken ti (~d,“,‘n) I’S where r/ is the difference heiween duplicates and n is the number of assays (15).
by epinephrine (Table 3) relca~e of clihydroxyacetone was similarly questionable while there was an almost tenfold increase in glycerol release. The finding of, at most, trace amounts of dihydroxyacetone in serum and in media used for incubation of nclil~osetissue is consistent with recent reports indicating that dihydroxyacetone levels are very low in tissues (11,12). Sensitivity
Using the mean of 0.418 and the standard error of the mean of 0.0045 (Table 1) the 95% confidence limits using deproteinized samples would be + 0.0106 pnole/ml. This would indicate that glycerol concentrations below about 0.011 pmoleJm1 would be indistinguishable from zero. This sensitivity is about half of that of Laurel1 and Tibbling’s fluorimetric method. Since the precision was better when removal of protein was not
Release
TABLE and I)ihydlox~-nretolle
of Glycerol
Sample Unstimulated adipose tissue Epinephrine st,imulated adipose
Number of samples
Mean glycerol release (pmoles,/g1
3 from
Adipose
Tkrue
llIcan dihydrosyacetone release (as gl!-cerol) (pmoler !g) 3 0.022
2 4
11.70
+ 0.286h
0. 0116 0.170 0.119 + O.WIY
III Vitroa
1 )ihydroxyaretolle release (t-l of glycerol release) 7 :: 11
a Pieces of adipose tissue (70-80 mg) pooled from the epididymal fat pads of four fed rats were incubated in Krebs-Ringer phosphate buffer with4:; bovine albumin (2.25 ml). After incubation for 1 hr the media were assayed for glycerol and dihydrosyacetone. * SEM. c Epinephrine cone was 1 pg/ml.
254
MOLLER
AND
ROOM1
required and since five times more sample could be used the lower for such samples would not be more than 0.002 ~moleJm1. Specificity
limits
and Interference
Table 4 lists compounds which were tested for interference with the assay of glycerol. Ethanol reduced NAD but to an insignificant degree as compared with glycerol. Dihydroxyacetone which is also phosphorylated by glycerol kinase (13) reduced NAD as expected. However, the yield was only about 60% of the theoretical (1 mole NAD reduced per mole dihydroxyacetone). The incomplete reaction with dihydroxyacetone is presumably due to a lower rate of phosphorylation (4). At the concentrations indicated in Table 4 dihydroxyacetone also slightly inhibited (less than 10%) the reduction of NAD by glycerol. PH Optimum reaction rat’es were achieved when the pH of the reagent mixture was adjusted t,o 9.2 as described under reagents. The final pH in the assay mixture under these circumstances was 8.65-8.70. A slightly higher pH (8.85) did not result in significant increases in reaction rate or final yield of NADH. At lower final pH values the yield of NADH decreased as indicated in Fig. 3. Effect of SW Groups The presence of an SH-compound dithiothreitol and mercaptoethanol
Various
Compounds
Final Compound Glucose Fructose n-glyceraldehyde Dihydroxyacetone Ethyleneglycol Ethanol Lactate P-Hydroxybutyrate Acetone a The decrease
Test,ed
is an absolute requirement. Cysteine, are all effective. Cysteine, which was
TABLE 4 for Int,erference
with
t,he Assay
of Glycerol
bM)
Reduction of NADH equiv t’o glycerol cone (mM)
Inhibition (at glycerol cone of 0.01 rnM)
1.69 1.69 0.02 0.02 0.60 16.90 3.38 3.38 3.38
0 0 0 0.006 0 0.001 0 0 0
0 0 0 slight* 0 0 0 0 0
cone in cuvette
degree of inhibition was not assessed of the combined recovery of glycerol
but’ was less than 10% and dihydroxyacetone
as indicated in a mixed
by the sample.
ENZYMATIC
ASSAT
FOR
!-A 75
8-O
255
GLYCEROL
8.5
9.0
PH
F’N. 3. lnfluencc of pH on reduction of NAD by plywrol. Glycerol was assayed as desrribrd under procedure esccpt that cystcine at a final concentration of 6.9 mM (triangles) was used in some espcrimcnts instead of mercaptoethanol (spheres). The pH values refer to the final pH in tllc, complete assay mixture.
used originally in these studies at, a final concentration of 6.9 mrq occasionally caused formation of a precipitate (probably of cystine) and at lower concentrations high blank reactions were obtained. This blank reaction was presumably due to the formation of a complex between NAD and cysteine (14). The blank reaction observed with mercaptoethanol, which can be used at a much lower concentration than cysteine, is probably due to a similar complex formation. Since the rate of complex formation increases rapidly between pH 8 and 10 (11) this reaction may place an upper limit on the pH in the assay. At the presently used mercaptoethanol concentration t’he blank reaction is constant and equivalent to about 2.5 X lo-” pmole glycerol in the assay. The catalytic system with albumin in samples was fully active for at least 2 hr since the appearance rates of NADH and recoveries of glycerol did not differ when identical samples of glycerol were added at rb~ hr intervals during this period to preincubated complete assay mixtures (results not shown). However, some batches of the mixed GDH and TIM preparation appeared to be labile \vhcn albumin was absent from samples.
The present method was compared with Wieland’s method (16) in four experiments. In one experiment aqueous solutions of glycerol were analyzed in triplicate by bot8h methods (Table 5). The regression lines were determined using the four points obtained by each analysis and estimated glycerol concentrations were derived from the regression line. The standard errors of estimate determined for each set of data indicate
256
Comparison
MOLLER
with
Wieland’s
Method
Wieland’s
AND
ROOM1
TABLE 5 and Analysis
of Glycerol
metshod
Standards
Present,
in Water
method
_. Glycerol cone (PM)
Optical density
Estimat,ed” couc (FM)
% of sample cone
Optical density
Estimat.ed” cone (GM)
:;, of sample cone
10 20 40 SO
0.014 0.029 0.061 0.121
9.3 19.3 40.7 so.7
93.0 96.5 101,s 100.9
0.042 0.083 0.161 0.320
10.4 20.5 39.9 79.2
104.0 102.5 99.x 99.0
Mean SEE” a Estimated b Standard
recovery: = +0.70
94.77,
from regression error of estimate.
Mean SEE”
recovery: = to.52
93.3yc
line.
that the present method in this experiment was somewhat more precise than Wieland’s method. Furt.her comparisons with Wieland’s method were made (Table 6) using samples obt,ained from incubations of isolated fat cells stimulated with epinephrine in Krebs-Ringer phosphate buffer. In the first experiment in Table 6, Wieland’s method was followed exactly as described in Ref. 16. In this experiment the mean glycerol concentration obtained
Comparison
with
TABLE 6 Wieland’s Met,hod and Analysis in Media Conkining Albumin Mean glycerol concentration b.Wml) ___-
+ SE&I (yO of mean)
Standards mean recovery (o/l)
s s
0.105 0.092
1.14 0.77
93.8 92.6
5 5
0.163 0.160
0.51 1.00
90.0 95.5
r
0.194 0.194
0.5s 0.97
97.7 95.1
Replicate samples Expt la Wieland’s method Present method Expt 2 Wieland’s method Present method Expt 3 Wieland’s method Present method
of (ilycerol
.G
a Samples analyzed were Krebs-Ringer phosphate albumin (4%) media separated from isolated rat, fat cells after one hour of incubat,ion with ephinephrine. The samples in all three experiments were analyzed by the present method wkhout deproteinizatinn. The samples were deprot,einized with perchloric acid in Expt 1, by ultrafiltration in Expt 2, and t.hey were not deproteinized in Expt 3 using Wieland’s method.
EXZYMATIC
ASSAY
FOR
GLYCEROL
257
by the present method was 87.6% of that obtained by Wieland’s method. However, the recoveries of glycerol in standards (made with KrcbsRinger phosphate buffer cont,aining 4% albumin) were nearly identical by the two methods. We thought on this basis that the higher values ohtained by Wieland’s method might be due to hydrolysis of glycerides during the precipitation of proteins with pcrchloric acid. To see whether this was the case samples from a second experiment and standards Fvere deproteinized by ultrafiltration through an Amicon PIZI-10 filter and analyzed by Wieland’s method. The results (Espt 2, Table 6) were nearly identical with the resulk obtained by the present method using non-deprot,einized samples. It nppcared, however, from t,hc recovery of glycerol in standards that there was a slight loss (appros 50/c of glycero1 in ultrafiltered samples. Since such a 10s~ ‘q might be concentration dependent the small difference between the results of the two methods could be due to variable degrees of losses in samples and rtandards. In the third experiment enzymatic analyses were therefore conducted direct,ly on non-clcprotcinizcd samples by both methods and identical results were obtained (Expt 3, Table 6). It would t8hus appear that Wieland’s method using dcproteinization with perchloric arid would tend to give too high values at least in samples obtained as described here. The two methods agrectl well on the other hand when precautions wcrc taken to minimize breakdown of glyccritlrs. In this comparison the present met,hod was more precise (and accurate‘) than the standard Wielnnd method (16) involving protein precipitation with perchloric acid. It would he expected that, the method in general would be more precise than methods yielding only 1 mole of NASH per mole of glycerol since the resuks by all methods for glyecrol rlcpcnd on differences betiveen initial optical densities before addit’ion of the enzyme specific for glycerol and the optican densities at t,he completion of t,he rcact’ion. This would bc particularly true of course for samples with low glycerol concentrations.
A new method for the analysis of glycerol is described. Employing a series of four enzymatic reactions its basic sensitivity is twice that. of existing enzymatic methods. In addition protein precipitation, when necessary, is possible in one step with tungstic acid. Dihydrosyacetone, which reacts incompletely in the assay, would have to he absent, from samples. This requirement was satisfied in samples ohtained from incubation of adipose tissue and in a clinical control serum. Although the reaction time is relatively lon,(r the method should he a useful epectrophotometric alternative to fluorimctrir methods where very low glycerol
258
MOLLER
AND
ROOM1
levels are encountered as, for example, in studies of basal (unstimulated) lipolysis when only small amounts of adipose tissue or small numbers of isolated fat cells are available. It would appear useful as well in analysis of tissue extracts for glycerol although the deproteinization required for such samples, because of the presence of various dehydrogenases and their substrates, would make it less sensitive than fluorimetric methods. ACKNOWLEDGMENT Financial support acknowledged.
from the Medical
Research Council
of Canada is gratefully
REFERENCES 1. WIELAND, 2. 3. 4.
5. 6. 7. 8.
0. (1957) Biochem. Z. 329, 313. HIMMS-HAGEN, J., AND HAGEN, P. B. (1962) Can. J. Biochem. Physiol. 40, 1129. KREUTZ, F. H. (1962) Klin. Wochenschr. 40, 362. GARLAND, P. B., AND RANDLE, P. J. (1962) Nature (London) 196, 987. LAURELL, S., AND TIBBLING, G. (1966) Clin. Chim. Acta 13, 317. SPINELLA, C. J., AND MAGER, M. (1966) J. Lipid Res. 7, 167. FARESE, G., AND MAGER, M. (1970) J. Lipid Res. 11, 274. VELICX, S. F. (1955) in Methods in Enzymology (Colowick, S. P., and Kaplan,
N. O., eds.), Vol. 1, p. 401. Academic Press, New York. 9. HORECKER, 13. L.. AND HORNBERG, A. (1948) J. Biol. Chem. 175, 385. 10. WIELAND, 0. (1965) irL Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), p. 244, Academic Press, New York. 11. WALTON, D. J.. AND GAUCHIE. L. M. (1972) Anal. Biochem. 46, 352. 12. CHARLTON, J. M., AND VAN H~YNINGEX, R. (1969) Anal. Biochem. 30, 313. 13. BERGMEYER, H. U., HOLZ, G.. KAUDER. E. M.. M~LLERING, H.. AND WIELAND, 0. (1961) Biochem. Z. 333, 471. 14. V.~N EYS, J., AND KAPLAN, N. 0. (1957) J. Biol. Chem. 228, 305. 15. HENRY, R. J. (1964) Clinical Chemistry. Principles and Technics, p. 125, Harper and Row, New York. 16. WIELAND, 0. (1965) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), p. 211, Academic Press, New York.