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A colorimetric method for estimating serum triglycerides
CLIKICA
CHIMICA
-1 COLORIMETRIC
ACTA
METHOD
393
FOR
ESTIMATING
SERUM
TRIGLYCERIDES
A calorimetric method for the estimation of serum glycerides is presented. The glycerol released by saponification of the serum glycerides is oxidized to formaldehyde which reacts with acetylacetone to form a yellow dihydrolutidine derivative absorbing at 405 m/l. The results correlate well with those given by the ZilversmitVan Handel method. Values for normal subjects range between 40~155 mg triolein per IOO ml (1.36-5.25 mequiv/l), the difference between the mean values for males and females being statistically significant by Student’s t test.
INTRODUCTIOX
Recent interest in atherosclerotic disease and its association with fat metabolismr-3 has caused an increasing demand in the clinical laboratory for the determination of serum triglycerides as well as those of cholesterol and phospholipids. In 1966, Kessler and Ledere? described a semi-automated fluorimetric procedure for quantitating serum triglycerides based on the Hantzsch condensation reaction between an amine, a p diketone and an aldehyde 5,6. Isopropanol extracts are prepared manually, the phospholipids are absorbed on Zeolite and the glycerides in the phospholipid-free extract are saponified to free glycerol. The glycerol is oxidized to formaldehyde which is then condensed with diacetylacetone and ammonia to give a fluorescent product, 3,5-diacetyl-1,4_dihydrolutidine. In the present communication the concentration of formaldehyde formed is determined by a calorimetric adaptation of the Hantzsch reaction as described by Nashs. MATERIALS
Ah’D METHOD
Reagents Zeolite m&we: Zeolite is obtained from Taylor Co., Baltimore, Md. The powder is ground in a Waring blender for 2 min and heated overnight at 110’. 50 g ground Zeolite is mixed with 5.0 g calcium hydroxide, 5.0 Lloyd’s reagent (hydrated alumiCliPI. CIzim. Acta, 22 (1g08)393-397
394
FLETCHER
nium silicate) and 2.5 g copper sulphate (Cu SO,.5H,O). The powder is mixed well before use. Potassium hydroxide. 5 9; KOH in isopropanol-water (40 : 60 v/v). Sodium nzetaperiodate: 0.025 M in 1.0 IV acetic acid. Working solution: dilute 12 ml of sodium metaperiodate and 20 ml isopropanol to IOO ml with 1.0 S acetic acid. This solution Acetylacetone:
must be freshly prepared daily. 0.75 ml 2,4-pentanedione and 2.5 ml isopropanol
in IOO ml 2 Jf ammonium acetate, pH 6.0. This reagent in a dark bottle and is stable for a month.
are dissolved
is stored in the refrigerator
l’rioleilz standard solution: Triolein (Applied Science Laboratories, State College, Pa.) (300 mg) is dissolved in IOO ml isopropanol. This standard solution is stab161 in the refrigerator indefinitely. Method The extraction
is carried out in Kimax
culture tubes, 16 x 150 mm with Teflon-
lined caps. Preparation
of the calibration cume standard solution is added to 8.8 ml isopropanol
0.2 ml Triolein
and I ml water,
A reagent blank is set up containing 9.0 ml isopropanol and 1.0 ml water. The solutions are mixed well and 2.0 g Zeolite mixture is added to each tube. The tubes are shaken on a mechanical shaker for IO min and then spun at 3500 rev./min for 5 min. The supernatants are decanted and may be stored in the refrigerator. From the supernatant, which contains 0.06 mg triolein per ml, a standard curve to match sera with 30-300 mg triolein per IOO ml is prepared. 0.2, 0.4, 0.8, 1.0, 1.4, 1.6 and 2.0 ml aliquots of the supernatant are diluted to 2.0 ml with isopropanol where necessary, corresponding to serum values of 30, 60, 120, 150, 210, 240 and 300 mg triolein per IOO ml. Each 2.o-ml portion of these dilute working standards and a 2.o-ml aliquot of the reagent blank is mixed with 0.6 ml 5 “/ KOH, the tubes stoppered and incubated at 60-70” for 15 min. After cooling 1.0 ml of the working sodium metaperiodate solution is added to each tube and the solutions mixed. 0.5 ml acetylacetone is then added, the solutions mixed, and the tubes stoppered and incubated at 50’ for 30 min. After cooling the color is read with an Eel Spectra 405 m/r, setting the instrument to zero with the reagent blank. Preparation
of semm
calorimeter
at
sanzples
0.2 ml serum is added slowly to 9.8 ml isopropanol.
nfter mixing 2.0 g Zeolitc mixture is added to each tube and the tubes shaken for IO min and then spun at 3500 rev./min for 5 min. The supernatants, as with the standard solution, may be stored in the refrigerator. e.o-ml aliquots are pipetted from each sample, 0.6 ml 5 ‘41 KOH added and tllcx procedure performed as described in the preparation of the calibration curve.
Factors
iq%lenci+zg the reaction Increase in the concentration
of potassium
hydroxide
above 5.00,$ or an in-
SERUM TRIGLYCERIDES COLORI~~~TR~ crease in the time of saponification solution
395
caused no further
of sodium metaperiodate
in I N acetic
release of glycerol.
acid was tested
The working
from 0.001 to 0.005
molarity and peak absorbance was found at a molarity of 0.003. Increase in the concentration of acetylacetone gave a higher absorbance of both test and reagent blank. The highest sensitivity with the lowest blank was found at a volume of 0.5 ml acetylacetone. If a serum or standard blank was prepared
in which the alkali and isopropanol
extract were not incubated at 60-70” but reacted immediately with the sodium metaperiodate and acetylacetone, the blank absorbance rose with the rise in glyceride concentration. Replacen~ent of the alkali with isopropanol gave values equivalent to the reagent blank indicating that formaldel~ydoge~~ic steroids and ~llonoglycerides were present in negligible amounts. It would seem that the presence of alkali allowed some saponification to occur during the period of color development at 50”.
the concentration
of triolein
405 m/l. is shown in Fig. I to be linear from 0-300
The relationship
between
mg triolein
and the absorbance
at
per IOO ml.
Recovery of glycerides Aliquots of triolein standard solution ranging in concentration from 30 to 150 mg per IOO ml were added to various serum samples and the glycerides estimated. The recovery ranged from g5-Io4s with a mean value of IOI y/din nineteen determinations. Recovery of glycerides was also evaluated by mixing different serum samples prior to the extraction. 96-104% recovery was found with a mean value of IOO 3; in six determinations.
The precision
of the method
was determined
by performing
twelve replicate
assays on two specimens, A and B. For A the values ranged from 116-131 mg triolein per IOO ml with a mean of IZZ mg per IOO ml & SD. 4.2 mg and a coefficient of variation 3.4 y/o.The values for B ranged from 62-72 mg triolein per IOO ml with a mean of 67 mg per 1;oo ml + SD. 3.0 mg and a coefficient of variation 4.5 y/o. A 0.4
-
0.3.
r/
0.2. 0.1 -
0
/
50
/
100 mg
P I I.50 triokin
200 per
250
300
lOOmi
Fig. I. Triolein concentratiotl and absorbance nt -105 mnjc. Fig. 2. Serum glycerides tiandel method.
determined
by the acetylacetonc
procedure and the Zilsersmit-Van
396
FLETCHER
Correlation zith the Zik~evsmit-l’ala Hawiel tecJalziqlle7 Glyceride estimation was carried out by the above method on 33 specimens in parallel with the method of Van Handel and Zilversmit as modified by Van Handel*. Close correlation (Fig. 2) was observed (Y r= o,()os, 1’ -< O.OOI).
Determinations were performed on sera drawn after an overnight fast from 20 healthy males and 26 healthy females ranging in age from IS to 49 years. The values for males showed a range of 50-155 mg triolein per 100 ml (1.09-5.25 mcquivll) \vitlr a mean of 99.0 I) SD. 30.5 mg triolein per roe ml (3.36 I- S.D. 1.03 mequiv/l) and for females of 40-11-j mg triolein per 100 ml (X.30-3.90 iuecluiv/l) with a mean of 75.4 ‘_ S.D. 23.9 nig triolein per 100 ml (2.55 1 S.D. o.S mequivl). The differenc.cL bctwccn the mean for males and females is significant, t == J.O, P c.- o.mr.
In the clinical laboratory glycerides have either been estimated indirectly frolll the difference between the serum concentration of the total esterified fatty acids ant1 the concentration of cholesterol and phospholipicl fatty acid esters” or by estimation of the glycerol portion of the glycerides. A disadvantage of the former method has been that the errors of the separate determinations are compounded in the final estimation of the glycerides. The most common determinations of the glycerol moiet\r have been based on the calorimetric reaction of formaldehyde with chromotropi;. acid’s”. The methods involve chloroform extraction of the lipids, evaporation of tlrc solvent before saponification, oxidation by sodium metaperiodate and a final co101 development with clu-omatropic acid at 100” for 30 min. In a recently. publishctl method by Sardesai and Manningl’ a similar extraction procedure was carried out but the formaldehyde was estimated calorimetrically by the Hantzsch reaction as in the abovc method. The procedure described in this paper differs from these calorimetric methods in several respects. By utilizing the extraction and chromatographic~ separation of lipids published by Kessler and Lederer* the necessity for evaporation of the solvent before saponification is avoided. Sodium metaperiodate, as a str-ong oxidizing agent, is known to interfere in the color development of formaldehyde wit11 either cllrorrlc~tropic acid or acetylacetone 10~3The excess periodate was removed in previous methods by the addition of sodium arsenite but it was found in this work that a low concentr-:Ition of sodium metaperiodatc would oxidize the glycerol without interfering in tlr(x color development. The normal range found in this study agrees well with the values of Carlson anti Wadstronl1o (21~135 mg/roo 1111)~and with those of Zilversmit and Van Handel (37-134 mg/'~oo nil)7 and a close correlation between the results by tfrc above proc(~dure and those of Zilversmit and Iran Handel was found. The difference betuw~n th(s means for males and females was significant and confirms the findings of Rcinhol(1 and coworkers” and Bucklev and coworkerslZ. The rise in the workmad in the clinical laboratory is being overcome by in creased automation and utilization of methods, such as the semi-automated fluorimetric technique for serum glycerides of Kessler and Ledere+. Since glyceride estima-
SERUM TRIGLYCERIDES
tion is important felt that
in physiological
the simple,
397
COLORIMETRY
reliable
and pathophysiological
and sensitive
method
conditions,
for the direct
the serum glyceride concentration presented above has a practical laboratory where such elaborate equipment may be lacking.
however,
it is
determination
of
value in a small
ACKNOWLEDGEMENTS
I wish to thank Dr. J. H. Wilkinson and Mr. I. Schreibman and encouragement during the course of this work.
for their advice
KEFEKENCES I 2 3 4
L. A. CARLSON, Acta Med. &and., 167 (1969) 339. P. T. Kuo, Med. Sci., 19 (1968) 46. A. KEYS, J. Am. Diatetic Assoc.. 50 (1967) 508.
Symposium, Automation in Analytical Chemisfr?~, G. KESSLER AND H. LEDERER, Technicoz New York, 1965, p. 341. 5 T. NASH, Biochem. J., 55 (1953) 416. 6 S. BELMAN, Anal. Chim. Acta, 29 (1963) 120. j E. V,w HANDELAND D. B. ZILVERSMIT,~. Lab. Clin. Med., 50(1959) 152. 8 E. VAN HANDEL, Clin. Chem., 7 (1961) 249. 9 J. G. REINHOLD,V. L.YONAN AND E. R. GERSHMAN, in D. SELIGSON(Ed.), Standard Methods ofCliGca1 Chemistry, Vol.4. Academic Press, New York, 1963. IO L. A. CARLSON AND L. H. WADSTROM,CZ~W. Chim. Acta, 4 (1959) 197. II V. &I. SARDESM AND J. A. MANNING, C‘lin. Chem..14 (1968) 156. 12 G. C. BUCKLEY, J.M. CUTLER AND J.A. LITTLE,Canad. Med. Assoc. J., 94 (1966) 886.
Clin. Chim. Acta,
22 (1968)
393-397
CHIMICA
-1 COLORIMETRIC
ACTA
METHOD
393
FOR
ESTIMATING
SERUM
TRIGLYCERIDES
A calorimetric method for the estimation of serum glycerides is presented. The glycerol released by saponification of the serum glycerides is oxidized to formaldehyde which reacts with acetylacetone to form a yellow dihydrolutidine derivative absorbing at 405 m/l. The results correlate well with those given by the ZilversmitVan Handel method. Values for normal subjects range between 40~155 mg triolein per IOO ml (1.36-5.25 mequiv/l), the difference between the mean values for males and females being statistically significant by Student’s t test.
INTRODUCTIOX
Recent interest in atherosclerotic disease and its association with fat metabolismr-3 has caused an increasing demand in the clinical laboratory for the determination of serum triglycerides as well as those of cholesterol and phospholipids. In 1966, Kessler and Ledere? described a semi-automated fluorimetric procedure for quantitating serum triglycerides based on the Hantzsch condensation reaction between an amine, a p diketone and an aldehyde 5,6. Isopropanol extracts are prepared manually, the phospholipids are absorbed on Zeolite and the glycerides in the phospholipid-free extract are saponified to free glycerol. The glycerol is oxidized to formaldehyde which is then condensed with diacetylacetone and ammonia to give a fluorescent product, 3,5-diacetyl-1,4_dihydrolutidine. In the present communication the concentration of formaldehyde formed is determined by a calorimetric adaptation of the Hantzsch reaction as described by Nashs. MATERIALS
Ah’D METHOD
Reagents Zeolite m&we: Zeolite is obtained from Taylor Co., Baltimore, Md. The powder is ground in a Waring blender for 2 min and heated overnight at 110’. 50 g ground Zeolite is mixed with 5.0 g calcium hydroxide, 5.0 Lloyd’s reagent (hydrated alumiCliPI. CIzim. Acta, 22 (1g08)393-397
394
FLETCHER
nium silicate) and 2.5 g copper sulphate (Cu SO,.5H,O). The powder is mixed well before use. Potassium hydroxide. 5 9; KOH in isopropanol-water (40 : 60 v/v). Sodium nzetaperiodate: 0.025 M in 1.0 IV acetic acid. Working solution: dilute 12 ml of sodium metaperiodate and 20 ml isopropanol to IOO ml with 1.0 S acetic acid. This solution Acetylacetone:
must be freshly prepared daily. 0.75 ml 2,4-pentanedione and 2.5 ml isopropanol
in IOO ml 2 Jf ammonium acetate, pH 6.0. This reagent in a dark bottle and is stable for a month.
are dissolved
is stored in the refrigerator
l’rioleilz standard solution: Triolein (Applied Science Laboratories, State College, Pa.) (300 mg) is dissolved in IOO ml isopropanol. This standard solution is stab161 in the refrigerator indefinitely. Method The extraction
is carried out in Kimax
culture tubes, 16 x 150 mm with Teflon-
lined caps. Preparation
of the calibration cume standard solution is added to 8.8 ml isopropanol
0.2 ml Triolein
and I ml water,
A reagent blank is set up containing 9.0 ml isopropanol and 1.0 ml water. The solutions are mixed well and 2.0 g Zeolite mixture is added to each tube. The tubes are shaken on a mechanical shaker for IO min and then spun at 3500 rev./min for 5 min. The supernatants are decanted and may be stored in the refrigerator. From the supernatant, which contains 0.06 mg triolein per ml, a standard curve to match sera with 30-300 mg triolein per IOO ml is prepared. 0.2, 0.4, 0.8, 1.0, 1.4, 1.6 and 2.0 ml aliquots of the supernatant are diluted to 2.0 ml with isopropanol where necessary, corresponding to serum values of 30, 60, 120, 150, 210, 240 and 300 mg triolein per IOO ml. Each 2.o-ml portion of these dilute working standards and a 2.o-ml aliquot of the reagent blank is mixed with 0.6 ml 5 “/ KOH, the tubes stoppered and incubated at 60-70” for 15 min. After cooling 1.0 ml of the working sodium metaperiodate solution is added to each tube and the solutions mixed. 0.5 ml acetylacetone is then added, the solutions mixed, and the tubes stoppered and incubated at 50’ for 30 min. After cooling the color is read with an Eel Spectra 405 m/r, setting the instrument to zero with the reagent blank. Preparation
of semm
calorimeter
at
sanzples
0.2 ml serum is added slowly to 9.8 ml isopropanol.
nfter mixing 2.0 g Zeolitc mixture is added to each tube and the tubes shaken for IO min and then spun at 3500 rev./min for 5 min. The supernatants, as with the standard solution, may be stored in the refrigerator. e.o-ml aliquots are pipetted from each sample, 0.6 ml 5 ‘41 KOH added and tllcx procedure performed as described in the preparation of the calibration curve.
Factors
iq%lenci+zg the reaction Increase in the concentration
of potassium
hydroxide
above 5.00,$ or an in-
SERUM TRIGLYCERIDES COLORI~~~TR~ crease in the time of saponification solution
395
caused no further
of sodium metaperiodate
in I N acetic
release of glycerol.
acid was tested
The working
from 0.001 to 0.005
molarity and peak absorbance was found at a molarity of 0.003. Increase in the concentration of acetylacetone gave a higher absorbance of both test and reagent blank. The highest sensitivity with the lowest blank was found at a volume of 0.5 ml acetylacetone. If a serum or standard blank was prepared
in which the alkali and isopropanol
extract were not incubated at 60-70” but reacted immediately with the sodium metaperiodate and acetylacetone, the blank absorbance rose with the rise in glyceride concentration. Replacen~ent of the alkali with isopropanol gave values equivalent to the reagent blank indicating that formaldel~ydoge~~ic steroids and ~llonoglycerides were present in negligible amounts. It would seem that the presence of alkali allowed some saponification to occur during the period of color development at 50”.
the concentration
of triolein
405 m/l. is shown in Fig. I to be linear from 0-300
The relationship
between
mg triolein
and the absorbance
at
per IOO ml.
Recovery of glycerides Aliquots of triolein standard solution ranging in concentration from 30 to 150 mg per IOO ml were added to various serum samples and the glycerides estimated. The recovery ranged from g5-Io4s with a mean value of IOI y/din nineteen determinations. Recovery of glycerides was also evaluated by mixing different serum samples prior to the extraction. 96-104% recovery was found with a mean value of IOO 3; in six determinations.
The precision
of the method
was determined
by performing
twelve replicate
assays on two specimens, A and B. For A the values ranged from 116-131 mg triolein per IOO ml with a mean of IZZ mg per IOO ml & SD. 4.2 mg and a coefficient of variation 3.4 y/o.The values for B ranged from 62-72 mg triolein per IOO ml with a mean of 67 mg per 1;oo ml + SD. 3.0 mg and a coefficient of variation 4.5 y/o. A 0.4
-
0.3.
r/
0.2. 0.1 -
0
/
50
/
100 mg
P I I.50 triokin
200 per
250
300
lOOmi
Fig. I. Triolein concentratiotl and absorbance nt -105 mnjc. Fig. 2. Serum glycerides tiandel method.
determined
by the acetylacetonc
procedure and the Zilsersmit-Van
396
FLETCHER
Correlation zith the Zik~evsmit-l’ala Hawiel tecJalziqlle7 Glyceride estimation was carried out by the above method on 33 specimens in parallel with the method of Van Handel and Zilversmit as modified by Van Handel*. Close correlation (Fig. 2) was observed (Y r= o,()os, 1’ -< O.OOI).
Determinations were performed on sera drawn after an overnight fast from 20 healthy males and 26 healthy females ranging in age from IS to 49 years. The values for males showed a range of 50-155 mg triolein per 100 ml (1.09-5.25 mcquivll) \vitlr a mean of 99.0 I) SD. 30.5 mg triolein per roe ml (3.36 I- S.D. 1.03 mequiv/l) and for females of 40-11-j mg triolein per 100 ml (X.30-3.90 iuecluiv/l) with a mean of 75.4 ‘_ S.D. 23.9 nig triolein per 100 ml (2.55 1 S.D. o.S mequivl). The differenc.cL bctwccn the mean for males and females is significant, t == J.O, P c.- o.mr.
In the clinical laboratory glycerides have either been estimated indirectly frolll the difference between the serum concentration of the total esterified fatty acids ant1 the concentration of cholesterol and phospholipicl fatty acid esters” or by estimation of the glycerol portion of the glycerides. A disadvantage of the former method has been that the errors of the separate determinations are compounded in the final estimation of the glycerides. The most common determinations of the glycerol moiet\r have been based on the calorimetric reaction of formaldehyde with chromotropi;. acid’s”. The methods involve chloroform extraction of the lipids, evaporation of tlrc solvent before saponification, oxidation by sodium metaperiodate and a final co101 development with clu-omatropic acid at 100” for 30 min. In a recently. publishctl method by Sardesai and Manningl’ a similar extraction procedure was carried out but the formaldehyde was estimated calorimetrically by the Hantzsch reaction as in the abovc method. The procedure described in this paper differs from these calorimetric methods in several respects. By utilizing the extraction and chromatographic~ separation of lipids published by Kessler and Lederer* the necessity for evaporation of the solvent before saponification is avoided. Sodium metaperiodate, as a str-ong oxidizing agent, is known to interfere in the color development of formaldehyde wit11 either cllrorrlc~tropic acid or acetylacetone 10~3The excess periodate was removed in previous methods by the addition of sodium arsenite but it was found in this work that a low concentr-:Ition of sodium metaperiodatc would oxidize the glycerol without interfering in tlr(x color development. The normal range found in this study agrees well with the values of Carlson anti Wadstronl1o (21~135 mg/roo 1111)~and with those of Zilversmit and Van Handel (37-134 mg/'~oo nil)7 and a close correlation between the results by tfrc above proc(~dure and those of Zilversmit and Iran Handel was found. The difference betuw~n th(s means for males and females was significant and confirms the findings of Rcinhol(1 and coworkers” and Bucklev and coworkerslZ. The rise in the workmad in the clinical laboratory is being overcome by in creased automation and utilization of methods, such as the semi-automated fluorimetric technique for serum glycerides of Kessler and Ledere+. Since glyceride estima-
SERUM TRIGLYCERIDES
tion is important felt that
in physiological
the simple,
397
COLORIMETRY
reliable
and pathophysiological
and sensitive
method
conditions,
for the direct
the serum glyceride concentration presented above has a practical laboratory where such elaborate equipment may be lacking.
however,
it is
determination
of
value in a small
ACKNOWLEDGEMENTS
I wish to thank Dr. J. H. Wilkinson and Mr. I. Schreibman and encouragement during the course of this work.
for their advice
KEFEKENCES I 2 3 4
L. A. CARLSON, Acta Med. &and., 167 (1969) 339. P. T. Kuo, Med. Sci., 19 (1968) 46. A. KEYS, J. Am. Diatetic Assoc.. 50 (1967) 508.
Symposium, Automation in Analytical Chemisfr?~, G. KESSLER AND H. LEDERER, Technicoz New York, 1965, p. 341. 5 T. NASH, Biochem. J., 55 (1953) 416. 6 S. BELMAN, Anal. Chim. Acta, 29 (1963) 120. j E. V,w HANDELAND D. B. ZILVERSMIT,~. Lab. Clin. Med., 50(1959) 152. 8 E. VAN HANDEL, Clin. Chem., 7 (1961) 249. 9 J. G. REINHOLD,V. L.YONAN AND E. R. GERSHMAN, in D. SELIGSON(Ed.), Standard Methods ofCliGca1 Chemistry, Vol.4. Academic Press, New York, 1963. IO L. A. CARLSON AND L. H. WADSTROM,CZ~W. Chim. Acta, 4 (1959) 197. II V. &I. SARDESM AND J. A. MANNING, C‘lin. Chem..14 (1968) 156. 12 G. C. BUCKLEY, J.M. CUTLER AND J.A. LITTLE,Canad. Med. Assoc. J., 94 (1966) 886.
Clin. Chim. Acta,
22 (1968)
393-397