The fluorometric assay of triglyceride by a semiautomated method

ANALYTICliL

BIOCHEMISTRY

The

25, 246-251

Fluorometric by

(1968)

Assay

a Semiautomated

Received

February

of Triglyceride Method1

5, 1968

In studies of the glucose-lipid interrelationship in women on oral st,eroidal cont’raceptiver, need arose for an accurate and rapid method for the assay of triglyceride in serum. For this purpose significant modifications were made in tl1is laboratory to an existing semiautomated method for the measurement of serum t,riglyceride (1). The method is based on the Hantzsch reaction in which diacetyldihydrolutidine is synthesized from formaldehyde and acetylacctone in the presence of excess ammonium ion. The reaction product is measured calorimetrically (2) or fluorometrically (3). Reduction of reagent volumes and concentrations to a workable minimum resulted in an improved flow pattern with good air segmentation and elimination of pulse. The “unsaponified blank” step was found to he unncce~Yary and watt omitted from the procedure, allowing an actual analysis rate of 40/hr on phospholipid-free rerm~l extracts. Results show a high degree of analytical precision and good yields from recovery experiments, which confirm the findings of the original authors.

Redistilled isopropnnol. General purpose grade isopropanol is refluxcd with 2,4dinitrophenylhydrazine and concentrated HCl for 2 hr and then redistilled. Silicic acid mixt’ure containing 200 gm silicic acid (Mallinckrodt) , 100 mesh, 10 gm CuSO, .5H,O, and 20 gm Ca (OH) ?. The silicic ncirl removes phospholipids and the copper and calcium salts eliminate glucose (4),

FLUOROMETRIC

ASSAY

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which can be readily oxidized to formaldehyde. These substances are ground in a mortar and the mixture is dried overnight at 110°C and subsequently stored in a desiccator.

1 mM solutions of trilaurin a,ntZ trimyristin (Eastman Kodak, used without~ further purification) in redistilled isopropanol. Dilutions made in iaopropanol to give a working range of 0.025-0.15 m&i’ are .;tandardized against. glycerol (British Standard 2621.25; 1955 App B). d2coholic KOH. Equal volumes of 2% aqueous KOH and redistilled isopropanol. So&~n p&otlate. 5 mM in 2 M acet.ic acid. Acetylacetone reagent. 3.5 ml of redistilled acetylacetone (Eastman Kodakj are made to 250 ml with 2 M ammonium acetate adjusted to pH 6 wit’h glacial acetic acid. It is important that this solution is made up freshly as required. The acctylacetone is stored at 4°C in a dark bottle. Sample preparation. 2 gm of the silicic acid mixture is added to Quickfit and Quartz glass-stoppered test tubes and mixed with 9.5 ml redistilled isopropanol: 0.5 ml serum is added from an Ostwald-Van Slyke pipet and mixed on a Vortex mixer for 15 sec. Mixing is repeated twice during a 20 min period. The tubes are then allowed to stand for 24 hr prior to centrifupation at’ 0°C in a refrigerated centrifuge at 2000 rpm for 10 min. The supernatants are t,ransferred to capped autoanalyzer cups for analysis.

-4 T(bchnicon AutoAnalyzer Sampler II, proportioning pump, and rariahlc-teml,erat~~re heating bath, a Locartc single-sided fluorometer Alark V fittecl with a flow cell, and a 10 mV potcntiometric recorder comprise the, instrumental system. Xo modification to any module is required except that a 5000 ohm resistance is inserted across the fluorometerrc~cortlcr outlet, Procedure The manifold and flow diagram are shown in Figure 1. The sample :tspirated at, 0.7 ml/mm, air at 2.0 ml, and alcoholic KOH at 1.2 ml/mm arc joined by a cactus before entering a single mising coil. Thiv airsegmented stream enters t’he lieatin, v bath, set at’ 58”C, in which hydrolysis is complrtcd during passage through a single 40’ delay coil. The hyclro1yzat.c is then joined by the rotlium periodate at 0.32 ml:min, foilowcd by the arctylacet~one reagent at 0.8 ml//min. The confluent streams are mixed in two tlouble mixing coils, then cntcr t’he ~eontl delay, roil of the

248

CRAMP

AND

ROBERTSON

heating bath for the oxidation and condensat,ion reactions. The stream finally enters the fluorometer flow cell after removal of air and is activated by ultraviolet light at 405 rnp. The increase of fluorescence is measured at 505 m,u. The lag time of t,he system is about 13 min. The samples are run at 40/hr with a 50% isopropanol wash using a 1:2 sample-to-wash ratio cam.

ml/m," ACETYLACETONE

’ DMC

DMC

I

/ / SMC

!SODIUM

0.8

PERIODATE

I !ISOPR~PANOLI

I I I I I

I

t

n

REAGENT

0 32

WATER

I

I

0

7s

l n

1ui In; I I I I n IWi

20

!AIR

,KOH I 1

REAGENT

1 2A

!SAMPLE

07s

s = Solvallex A = AcIdflex FLUORIMETER

FIG,

1. Flow diagram

for automated

RESULTS

AND

fluoromekic

triglyceride

method.

DISCT’SSION

The continuous-flow system performs t’hree discrete reactions on the serum extract: hydrolysis of the triglyceride to glycerol; oxidation of the glycerol moiety to formaldehyde; and final production of a stoichiometric amount of diacetyldihydrolutidine, which is measured fluoromctrically. 1. Hydrolysis of the triglyceride with alcoholic KOH. The alcoholic base is essential for complete saponification of the triglyceritle (5) ; 1% KOH in a solution of 50% isopropanol and water was found to be optimal to effect complete hydrolysis: glycerol production is no longer quantitative at a lower solvent concentration. 2. Oxidation of the glycerol moiety to formaldehyde with sodium periodate. A 5 mM solution in 2M acetic acid pumped at 0.32 ml/min is about ten time5 the absolut’e stoichiomctric rcqnirctncnt I 6 I .

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TRIGLYCERIDE

3. Formation oj the flzcorophor. This was carried out essentially according to Belman (3). Acetylacetone is dissolved in 2 M ammonium acetate, pH 6, and the reaction performed in a constant-temperature oil bath at 58°C. We found, however, that the sensitivity of the reaction could be considerably enhanced by increasing the molarity of the acetylacetone: a 7-fold increase in acetylacetone concentration increased background fluoresceiice by only 5%. We have omitted the “unsaponified” blanks described in the original m&hod, obtained by bypassing the heating bath during the initial mixing with alcoholic KOH. These were found to be negligible unless: (I) glucose was incompletely eliminated from the extracts and (a) serumfree glycerol levels were raised above a level of about 200 jLmoles/liter [normal levels 48 k 4 ,umoles/liter; diahet,ic levels 90 r+ 6 ,Imoles/liter (7) I. Under our esperimental conditions the quantities of copper sulfate and lime in the extraction mixture do not effectively remove glucose unless 241

!40

160

60

40

c

2. Recording showing calibration curve with 40, 80, 160, and 240 mg/lOO ml standard peaks, and reproducibility of duplicates of standards. FIG.

250

CRA.\IP

.4XD

ROBERTSON

the serum/extraction mixture is left to stand for at least 24 hr prior to centrifugation and analysis. Attempts to increase the copper and calcium salts up to levels recommended by Van Slyke (4) caused t.urbidity due to colloidal suspensions. Glycerophosphate and glyceraldehydc 3-phosphate did not interfere in the reaction up to concentrations of 1 mM.

FIG. manual

3. Scattergraph and fluorometric

of correlation between results methods. The line represents

obtained z = y.

from

129 scra,

by thri

Under the conditions described a straight-line relationship was obtained with standard t,riglyceride solutions in the range 0.025 to 0.25 mM, equivalent to a serum triglyceride range of 0.5 to 5 mM or 40 to 400 mg/lOO ml. (In this laboratory the factor 80 is used to convert, millimole.. per liter to milligrams per 100 ml.) Greater precision in int.erpreting th:. lower concentrat,ion range was obtained by incrcaxing the sensitivity to

FLUOROMETRIC

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allow full-scale deflection of the recorder with a 0.15 mM triglyceride standard solution. The precision of the met.hod is 2.2 mg over the range 40-250 mg per 100 ml. This value is the standard deviation(s) of 50 duplicated samples, obtained by applying the formula

where rE is the difference between duplicate samples and N the number of duplicate pairs. Standardized serum extracts are included in each batch of analyses as a routine check of precision. The mean of 18 recovery experiments in which standard amounts of triglyceride were added to serum extracts was 99.6% (range 94-106%). 129 serum samples were assayed and compared with results obtained by a manual modification of the van Handel and Zilversmit technique (8). The distribution and regression equations are shown in Figure 2. The correlation coefficient is r = 0.98 (P < 0.001). The respective mean values obtained by the manual and automated methods were 114 and 112 mg/lOO ml (Fig. 3). An analysis of variance revealed that the variation between the two methods was not significantly different from the variance within each method. The method as described is suitable for routine or research assay of triglyceride, and has proved reliable, precise, and economical during many months of constant use in this laboratory. ACKNOWLEDGMENT We wish to thank Dr. Victor Wynn, Reader in Human Metabolism in the University of London, for providing facilities and opportunity to carry out this work. REFERENCES

G., AND LEDERER, H., Technicon Sy.mposium, “Automation in Analytical Chemistry,” Mediad, Inc., New York, 1966. 2. KASH, T., Biochem J. 55, 416 (1953). 3. BELMAN, S., Anal. C&m. Acta 29, 120 (1963). 4. VAN SLYKE, D. D., J. Biol. Chem. 32, 455 (1917). 5. HENRIQUES, R., Z. Angew. Chem. 24, 721 (1895). 6. VORIS, L., ELLIS, G., AND MAYNARD, L. A., J. Biol. Chem. 133, 491 (1940). 7. GARLAND, P. B., AND RANDLE, P. J., Nature (London) 196, 987 (1962). 8. YIN H.~NDEL, E., AND ZILVERSMIT, D. B., J. Lab. C&z. Med. 50, 152 (1957). 1. KESSLER,