The determination of fructose in biological fluids using anthrone

BRIEF

NOTES

"67

The determination The method

of fructose in biological fluids using anthrone

described

here is a modification

Handel1 and which has been adapted

of the anthrone

for the deterlninatioll

method

of fructose

of Van

in biological

fluids. METHOD

Dissolve 150 mg anthrone in x00 ml of SO::) v/v sulphuric glass bottle and make up fresh each week.

acid. Store in dark

(I) Pipette 0.5 ml volumes of fructose standards, water (blank) known deproteinized samples into a series of Isox 16 nlm test tubes.

and the un-

A suitable set of standard fructose solutions contains 2.5, 5, IO and 15 mg/~oo ml (growth of microorganisms is prevented by the use of a saturated solution of benzoic acid as the dilutant). Ueproteinization of the biological fluid can conveniently be achieved by taking I volume of the biological fluid +5 volumes of water + z volumes ~.zqb NaOH + z volumes 5’:;’ ZnSO,; the deproteinized filtrate is therefore a I :IO dilution of the original fluid. (2) Run in 3 ml of the anthrone reagent into each tube from a burette and shake well. (3) Incubate 35--37’

all tubes in a thermostatically

for 30 min. (1) Read absnrbances

of the green-blue

controlled

water bath at between

solution in suitable

calorimeter

{Spek-

ker al~sorptiolneter-~ilger & Q’atts, Ltd. London) against the water blank using celis with an 0.5~cm light path length and orange filters (600 mp-Ilford 607). A linear relationship of absorbance to fructose concentration over the range o-15 mg/roo ml (Fig. I).

O0

5

IO

is obtained

when examined

15

CGNCE~TRATlG~-m~/lOOm~

Fig. I. The Iinear development of colour with increasing concentrations of fructose (0) and glucose (,-). The absorbance was read in o.s-cm cells after the incubation of 0.5-ml volumes with 3 mi of the anthrone reagent for 30 min at 35’. Clin.Chim. Acta, 26 (1969) 167-1G9

168

BRIEF

The absorbances

for the corresponding

glucose

concentrations

SOl‘liS

are extremeI>

low (Fig. I).

Table I shows the recovery of fructose when added to adult sheep plasma in the presence of varying concentrations of glucose. The values obtained by the anthrone method were good and superior to those given by a resorcinol method. Agreement between duplicate determinations was considerabl!. better with the anthrone method.

A number

of carbohydrates

were tested for their reactivity

with the anthrone

reagent under the conditions described here. Apart from sorbose and those substances containing a fructose moiety the only carbohydrates tested which showed gross reactivity were the pentoses (Table II).

The method has been used to determine the fructose concentration in foetal sheep plasma andin the plasma of adult sheep in whom a fructosaemia was established. A total of 414 samples of plasma were estimated for fructose both by the anthrone method and by the resorcinol method of Chinard et aL3. The overall mean values for the fructose concentration in these samples were 75 and 72 mg/roo ml for the anthrone C&n. Chzn?. Acta,

26 (1969) 167-169

BRIEF

169

NOTES

and resorcinol methods respectively. The reagent fructose and inulin concentrations in urine. I should like to thank Department

St. Magi’s

Mrs. E. Fenton

may

also be used to determine

for her assistance.

ofPhJlsiology, Hospital Medical School,

D. A. NIXOX

London, II’. 7 (L’.K.) I 13. \..a ~LISDEL, Axal. l3iochcm., 19 (1967) 93. 2 X. ST. G. HUGGETT AND I). A. NIXON, I.avzcet, ii (1957) 36X. A. ST. G. HUGGETT, 3 1;. P. (‘HINARD, 1'. L)ANESINO, \v. L. HARTMANN, Ibun-OLDS, J. Physzol., 132 (1956) 289.

Received

11'. PAUL

.4x13 S. K.

hr.

April 18, 1969 Cliz.

Chin?. Actn, 26 (1969)

167-169