BIOCHEMICAL MEDICINE 9, 62-72 ( 1974)

Interference by Myo-lnositol of the Biuret Reaction

in the Ultraviolet Range1

W. BURG1 AND H. KAUFMANN

Department of Clinical Chemistry, Kantonsspital, 5001 Aarau, Switzerland

Received April 3, 1973

For the determination of total protein in biological fluids of low protein concentration, a variety of methods are employed. A widely accepted procedure is that of Lowry (l), using the phenolic reagent of Folin and Ciocalteu (2). Nonlinearity of the standard curve and differences for the type of protein analyzed are serious disadvantages. The modification proposed by Rieder corrects for the interference due to paraamino- salicylic acid (3). In many laboratories total cerebrospinal fluid (CSF) protein is estimated ,by turbidimetric measurements which, however, are known to lack reliability (4). Th e most generally accepted procedure for quantitation of protein is the biuret method. In order to overcome the drawbacks encountered with its low sensitivity, several authors have reported modifications for estimating protein in low concentrations including CSF (5-9). Although interference by substances other than those containing peptide linkages is known ( 10, ll), the specificity of the biuret reaction has been considered to be sufficient.

The present paper describes the interference of the biuret reaction by a substance, which is physiologically ,present in biological fluids and tissues and which has been identified as myo-inositol.

MATERIAL AND METHODS

The cerebrospinal fluid specimens were received for routine analyses from patients of the medical and pediatric departments. The fluid was drawn by lumbar puncture, excepting the newborn and premature babies, who were subjected to suboccipital puncture. No difference was made in the subsequent processing between the two different kinds of samples. The aliquots not used for routine analyses were immediately separated from the cellular elements by centrifugation and stored at -18°C until used.

‘A preliminary note of this work has been published in Z. Klin. Chem. I&n. Biochem. 10, 432, 1973.

62

Copyright @ 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.

MYO-INOSITOL AND BIURET REACTION 63

The protein content was determined by two methods. Method 1. To 0.5 ml of biuret reagent, 0.1 ml of specimen to be ana-

lyzed is added. A blank containing distilled water instead of spinal fluid is prepared accordingly. After development of the biuret complex, the optical densities are read at 334 nm against the reagent blank. The details of this procedure have been published elsewhere (9).

Alethod 2. From a l.O-ml sample, the protein is precipitated with perchloric acid. The precipitate is dissolved with the biuret reagent and the absorbancy of the color complex is measured at 546 nm (8). The photometric readings at 334 and 546 nm were carried out in an Eppendorf photometer equipped with a mercury vapor lamp and a photomultiplier. The spectral curves were obtained in a Zeiss PM 4 spectral photometer with a grating monochromator.

Ultrafiltration

For the filtration experiments, an AMICON 12 ultrafiltration cell and SARTORIUS SM 12136 membranes were used.

Thin-Layer Chromatography

Readymade glassplates “Merck” Silica gel F 254 were used throughout. Solvents. ( 1) pyridine : ethylacetate : glacial acetic acid : water = 36 : 36 :

7 : 21 (v/v) ; (2) ethylacetate : glacial acetic acid: methanol : water = 60 : 15:15:10 (v/v); and (3) n-butanol : pyridine : water = 75 : 15: 10 (v/v).

Staining solutions. ( 1) coned sulfuric acid: glacial acetic acid = 1: 1 (v/v) ; (2) silver nitrate-2 N sodium hydroxide ( 12) ; and ( 3) the GOD/ POD reaction was used as a specific stain for glucose according to the manufacturers direction ( 13).

High Voltage Electrophoreti

A Camag HVE cell No. 61000 was employed. Electrophoresis was carried out in a 0.05 M Na-tetraborate buffer, pH 9.2 at 75 V/cm for 45 min of the following carbohydrates: fructose, glucose, galactose, maltose, mannose, lactose, ribose, xylose, and myo-inositol.

Column Chromatography. The procedure was carried out on Dowex 2 X10, 100-200 mesh, acetate form and Dowex 50 X2, 200-400 mesh, H+ form. The resins were recycled before use. Columns, 1.2 cm in diameter, were packed to a height of 3 cm with Dowex 2 and this was overlayered by the same volume of Dowex 50, both in 0.5 M acetic acid. Sampl:s were applied and eluted with the same solvent. Eluates were analyzed by the biuret Method 1.

64 BiiRGI AND KAUFMANN

Preparation of Myo-lnositol Hexaacetate

Three hundred and eighty milligrams of myo-inositol were dissolved in 15 ml acetylating reagent, consisting of a 1: 2 mixture of acetic acid anhydride and pyridine. The solution was allowed to stand at 25°C for 24 hr. The reaction mixture was evaporated to dryness and dissolved in 50 ml of ethylacetate, then the solution was washed three times with 10 ml of 5% NaHCO.,, dried over Na$O,, and evaporated to dryness. The residue was dissolved in boiling ethylacetate and allowed to crystallize.

Isolation of the Interfering Substance

Preliminary Experiments. From sample No. 14, the ultrafiltrate was passed through a Dowex 20 H- and a Dowex 50 H+ column. The eluate was concentrated and subjected to thin-layer chromatography. Separation into two spots was achieved in silica gel with solvent No. 1. Both spots gave a positive reaction with AgNO,-NaOH and the larger one with the higher Rf value could be identified as glucose with stain No. 3. Column chromatography on Dowex 2, borate form, was unsuccessful in separating off glucose, since the unknown substance formed a borate complex as well.

Isolation and Zdentijication of Free Myo-lnositol

The starting material consisted of 18 ml pooled cerebrospinal fluid of premature babies. After adjustment of the pH to 4.5 with 1 N acetic acid, 80 ml of absolute ethanol were added dropwise under constant stirring. The solution was kept at 5°C overnight. The protein precipitate was centrifuged off and the supernatant passed through a Whatman No. 1 filter paper. The filtrate was evaporated to dryness and the residue dis- solved in 15 ml distilled water. From this solution, the lipids were ex- tracted five times with 5 ml of ether. The extracts were discarded. The aqueous phase was evaporated to dryness and dissolved in 3.9 ml distilled water. This solution was desalted in the Dowex mixed-bed column. The fractions of the eluates giving a positive reaction at 334 nm with the biuret reagent were combined, evaporated to dryness, and dissolved in 1.0 ml distilled water. Thin-layer chromatography and high-voltage electrophoresis demonstrated the presence of two components, i.e., glucose and myo-inositol.

The separation of myo-inositol from glucose was achieved by prepa- rative thin-layer chromatography. Two silica gel plates F 254 (Merck 57150025) were charged each with 0.5 ml on a E-cm-long application line. After a running time of 4 hr in solvent No. 3, the plates were dried

MYO-INOSITOL AND BIUFET REACTION 65

and a small band on the sides of each plate was stained with AgNO,- NaOH. The fraction corresponding to myo-inositol was scraped off the plates and extracted three times, each of which for 1 hr with 4 ml of 10% ethanol. The combined extracts were dried. On a subsequent thin-layer chromatographic and high-voltage electrophoretic analysis, only one AgNO,-NaOH-positive spot could be detected, indicating that glucose had been separated off. The extract was dried to completeness over P,O,. To the residue, 6 ml aeetylating agent were added and the solution was stirred for 48 hr at room temperature. The dried hexaacetate was extracted three times with 2 ml hot ethylacetate and the combined extracts were washed twice with 2 ml 1% NaHCO, and once with 2 ml H,O. The organic phase was dried and the resulting brown residue was sublimated at 12 mmHg and at 160°C. The sublimate had a yellow color. It was recrystallized twice from ether. The yield amounted to 0.6 mg of colorless crystals which had a melting point of 211°C. The reference substance, myo-inositol, which was acetylated by the procedure as described above, showed a melting point of 213°C.

Gas chromatography was carried out in a Perkin-Elmer model 900 ap- paratus, equipped with a flame ionization detector and a 200-cm column of 2.2 mm i.d. packed with 5% SE 30 on gaschrom Q W-100. The temper- ature of the FID was set at 300°C. Helium was used as carrier gas with a flow rate of 30 ml per minute. The injection block had a temperature of 300°C and the experiments were run at a column temperature of 180°C. The peaks corresponding to myo-inositol were analyzed in a MS-9 HE1 Manchester mass spectrograph. The conditions were a ionization potential of 70 V and a source temperature of 250°C.

RESULTS

The quantitation of CSF protein of premature and newborn babies and of an ad& male by the two different biuret procedures reveals dif- ferences as indicated in Table 1. In most samples analyzed, the values obtained by Method 1 are considered to be erroneously high, since in no instance could they be correlated with the clinical condition. Figure 1 represents the spectral absorbance curves of two ultrafiltered spinal fluid samples, one derived from a 47-year-old patient, the other from a S-week- old baby. Both curves are identical with the reagent blank from 420 nm upward. In the uv-region, however, the ultrafiltrate of the newborn’s spinal fluid shows an absorption which increases with decreasing wave- length. The spectral behaviour of the ultrafiltrate of sample No. 14 (Table 1) indicated an interference of the biuret reaction by a small molecular- weight substance present in the cerebrospinal fluid of this patient. The information obtained from the preIiminary experiments suggested that

66 BihGI AND XAUFMANN

TABLE 1

RESULTS OF CSF PROTEIN L)E:TERMIS.\TIONS my Two MODIFKATIONS OF THK

BIURET METHOD: Method 1, I)IRISCT DETERMINATION, OMITTING

I~ICPROTEINIZATION FOLLOWI’;D HY PHOTOMETRIC RE.ZDINGS .xl

334 NM. Method 2, PHF;CIPIT.ZTION OF THE PHOTICIN HE- HCIOa PRIOV TO h! ! : \cT7’11~:MI:ST OF Ansor::c.\~vc~~ .\I’ 346 x11

Sample no. Term of birth

Age at time of pnp!ys:s

Protein concentration fmg/lOO ml)

1

2

3

4 5

6

7 8

9 10

11

12 13

14

1 .i

Full term

Full term Full t,erm

Full term Full term

29 Weeks 36 Weeks

33 Weeks

Full term Full term

Full term

34 Weeks Full term

3 Days

10 Weeks

2 Years

5 Weeks

10 Years

4 Weeks

X Weeks

6 Weeks

4 Weeks

3 Weeks

2 Days

4 Weeks

4 Weeks

5 Weeks

47 Years

316 84

156 s4

54 26

193 63 2. ‘3 15

281 111

186 79

157 56

244 32

274 162

394 86

253 58 ‘222 79

166 X5

75 73

wave length

FIG. 1. Spectral absorption curves of biuret complexes obtained by Method 1, derived from ultrafiltrated CSF of an adult patient (-O---) and of a newborn

baby (-a--). They correspond to No. 15 and No. 14, respectively, in Table 1. The third curve (---) represents the reagent blank. The photometric readings were performed against distilled water,

MYO-INOSITOL AND BIURET REACTION 67

this substance perhaps has a polyhydroxy type of structure, since it ap- peared to form a borate complex on column chromatography similar to glucose. Moreover, the electrophoretic mobility at pH 9.2 in borate buffer of the unknown was identical with that of myo-inositol. Identity was confirmed by analysis of the properties of the hexaacetate derivative. The melting points of the hexaacetates of the unknown and of reference myo- inositol were found to be almost identical and no depression was ob- served after mixing the two samples. Definite proof of identity was demonstrated by gas chromatographic and mass spectrographic analysis of the unknown and of the reference compound.

Some sugars were tested by Method 1 in order to find other sources of interference. They were dissolved in distilled water to a concentration of 10 mmoles/liter. The results of these experiments are recorded in Table 2. They show that in addition to myo-inositol, mannose at a con- centration of 180 mg/lOD ml forms a complex with the biuret reagent, resulting in an optical density of 1.0 at 334 nm. The biuret complex produced by ribose absorbs approximately one third of the light at this wavelength as compared to myo-inositol. Mannose and ribose, however, could not be detected in CSF by high-voltage electrophoresis in those samples which ‘contained myo-inositol.

The absorption spectrum of a S-mM aqueous solution of myo-inositol treated by biuret method 1 is recorded in Fig. 2. The curve shows a definite peak at 300 nm. There is absolutely no absorption above 480 nm. It should be noted that myo-inositol itself absorbs no light at the wave- lengths tested.

TABLE 3

RELCTION OF SOME SUGARS JVITH THE BIURET RLIGENT. SOLUTIONS,

CONTAINING 10 mmoles/l WERE ASSAYED ACCORDIXG

TO Method 1 (SEE TXXT)

sugar Optical densit,y at 334 nm

Arabinose 0.046

Ascorbic acid -0

Fructose 0.078

Glucose 0.040 Galactose 0.0%

Maltose 0.038 Mannose 1.010 Myo-inositol 1.840 Lactose 0.086

Ribose 0.604 Sylose 0.033

0 The biuret reagent is reduced to free copper.

68 BijRGI AND KAUFMANN

wave length

FIG. 2. Absorption spectrum of the complex formed by myo-inositol and the

biuret reagent according to Method 1. The sample concentration was 5 mM in

distilled water. The photometric readings were made against the biuret reagent.

The recovery of myo-inositol after ethanol precipitation, column chro- matography, and preparative thin-layer chromatography amounts to 79% as measured by the optical density of the biuret color at 334 nm. The losses during the isolation are shown in Table 3. Most of the loss is observed during the desalting procedure on Dowex.

Gas chromatography and mass spectrography demonstrated identity with the reference compound of the hexaacetate sample derived from pooled cerebrospinal fluid of premature and newborn babies.

DISCUSSION

Photometric readings of the purple biuret complex are usually carried out in the visible range of the light spectrum. The major drawback of

TABLE 3 RECOVERY OF THE NONPRECIPITAI(LE BIURET COLOR OF POOLED CSF. SINCE

THE VALUE OF THE STARTING MATERIAL INCLUDES THE BIUR~T COLOR PRODUCED BY THE PROTEIN, THE OPTICAL DENSITY READ FOLLOWING

ETHANOL PRECIPITATION WAS TAKEN AS 100yO

Total OD 334 nm Recovery

Step 1 Step 2 Step 3

Starting material

After ethanol precipitation After desalting on Dowex After elution from tic

25.2

1.5.3 100% 13.0 85% 12.0 79yc

MYO-INOSITOL AND BIUBET REACHOK 69

this procedure is its relatively low sensitivity. In the presence of low

protein concentration a large sample size is necessary from which the protein is concentrated by precipitation prior to the addition of the biuret reagent. This prerequisite applies in particular to the determination of total protein in cerebrospinal fluid (CSF), urine, and other biological fluids.

The sensitivity of the biuret reaction, however, is markedly increased when advantage is taken of the high spectral absorbance of the color complex in the near uv range. Thereby, the disadvantage mentioned before can be circumvented. Thus, Itzhaki and Gill (7) described a method for estimating proteins with the biuret reaction and measuring the optical density at 310 nm. These authors found this procedure td be both simple and sensitive, since a &fold increase in sensitivity over the ordinary biuret method is gained. Moreover, their results demonstrated a fair nonspecificity for the type of protein investigated. In particular, high concentrations of DNA did not intedere, rendering it suitable for quantitation of enzymes and other proteins in tissue extracts.

Based 011 the same principle, Biirgi and coworkers (9) published a procedure for the determination of total protein in cerebrospinal fluid, which requires only 0.1 ml of sample. Using a modified biuret reagent, i.e., copper acetate instead of copper sulfate, the concentration of NaOH could be decreased to 3%. To prevent turbidity, ethylene glycol was added as a complexing agent. The photometric readings were performed in an Eppendorf photometer at a wavelength of 334 nm. This modification of the original biuret method proved to be valuable in clinical routine work. However, it became evident that false high values were encountered which could be related to patients of the pediatric department and in particular to premature and newborn babies. Since errors of specimen collection could be excluded it had to be assumed that factors intimately related to the chemistry of the reaction with newborn or premature infant CSF are responsible for this interfering phenomenon.

The spectral curve of the product of the biuret reagent and of a typical CSF giving a false high value suggested the presence of a substance form- ing a complex with copper in alkaline solution. This complex apparently absorbs only uv light, but no absorption is recorded in the visible range, or at 545 nm where the biuret complex reaches its peak. The chromato- graphic behaviour of the unknown compound was similar to that of glucose, both forming a borate complex. The separation from glucose by thin-layer chromatography yielded a pure substance which, by means of high-voltage electrophoresis and thin-layer chromatography, was tentatively identified as myo-inositol. Determination of the melting point of the hexaacetate of the unknown and of myo-inositol hexaacetate

70 BtiCI AND KAUFMANN

sample followed by gas chromatographic and mass spectrographic analy- sis revealed that these two derivatives are identical.

Interference of the biuret reaction by certain nonprotein compounds has been reported. Robson et al. (10) p resented results demonstrating absorption of Tris buffer at 540 nm. Recently, these findings have been confirmed by Sokolowski (11) who found in addition an interference by triethanol amine buffer. A positive biuret reaction with reducing agents such as 2-mercaptoethanol is mentioned in the work of Bennet (14). However, myo-inositol hitherto was not known to react with the biuret reagent. This may, in part, be due to the fact that absorption measurements of this method have primarily been limited to visible wavelengths.

Myo-inositol is widely distributed in nature. It occurs either free or esterified to form inositol-phosphatides. In an extensive survey, Beckman and Petith (15) summarized the state of knowledge up to 1970. It may be noted that the normal concentration of free myo-inositol in humans ranges between 0.5 and 1.0 mg/lOO ml in blood serum (16), 2.0-3.4 mg/ 100 ml in CSF ( 17), and in urine S-144 mg/24 hr ( 18). These levels fluctuate with a number of disease states.

The absorption spectrum of the complex between alkaline copper and myo-inositol is limited primarily to the ultraviolet range. Absorption begins at 480 nm and increases with decreasing wavelengths, reaching a distinct peak at 300 nm. As demonstrated in Fig. 2 the extinction of the inositol biuret complex at 334 nm reaches approximately two-thirds of this peak value at 300 nm. The aqueous solution of inositol with a con- centration of 180 mg/lOO ml shows an absorbance of 1.84 which cor- responds, according to the modified biuret Method 1, to a protein equivalent of 800 mg/lOO ml. The values obtained by the two methods (Table 1) differ up to 300 mg/lOO ml from each other. In experiments to be reported later it has been shown that this difference is equivalent to approximately 3 mmoles/liter CSF inositol (54 mg/ 100 ml). This con- centration would explain a difference in total protein of 300 mg between the two biuret methods due to myo-inositol in the stated concentration. Although the normal concentration of free inositol is reported to total only 2.0-3.4 mg/lOO ml CSF and no values are available for the human newborn, it is interesting to note that the concentrations of CSF myo- inositol in the fetus of the goat and sheep are as high as 55 mg/lOO ml and 140 mg/ 100 ml, respectively ( 19).

It must be added that the interference of the biuret reaction by sub- stances other than myo-inositol cannot be excluded, when fluids such as CSF are analyzed.

As represented in Table 2, a number of sugars give a positive reaction

MYO-INOSITOL AND BIURET REACI’IOiX 71

at 344 nm. It is unlikely, however, that these compounds occur in concen- trations high enough to interfere to a significant extent with the biuret reaction. In spite of the fact that, according to Table 3, a recovery of approximatively 79% in optical density was obtained. other compounds present in CSF not mentioned here could absorb light in the uv region as a copper complex.

The findings presented in this paper indicate that the determination of total CSF protein by the biuret method requires separation of low mole- cular-weight interfering substances prior to the development of the biuret reaction. This precaution to avoid false high total protein values applies to urine, other biological fluids of low protein concentrations, and tissue extracts as well. This precaution has to be kept in mind especially in those cases in which the myo-inositol concentration is known to be elevated.

SUMMARY

The interference of the biuret reaction in the ultraviolet range by a substance physiologically present in cerebrospinal fluid is reported. After precipitation of the protein by ethanol and desalting by column chro- matography, this substance was isolated from pooled CSF of premature babies and newborn by thin-layer chromatography. The hexaacetate derivative of the unknown and of reference myo-inositol revealed identity as judged by melting point determinations, gas chromatographic and mass spectrographic analyses.

It is concluded that precipitation of the protein from dilute solutions is a prerequisite for the quantitation by the biuret reaction in the ultra- violet range.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the gas chromatographic and mass spectro- graphic analyses carried out by Drs. A. Lampart and W. Vetter, F. Hoffmann-La

Roche, Basel.

REFERENCES

1. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Viol. Chem. 193,265 ( 1951).

2. MEITES, S., (Ed.), “Standard Methods of Clinical Chemistry,” Vol. 5, p, 223. Academic Press, New York, 1965.

3. RIEDER, H. P., Clin. Chim. Acta 6, 671 ( 1961).

4. LUPS, S., AND HAAN, A. M. F. H., “The Cerebrospinal Fluid,” p. 297-311. Elsevier, New York, 1954.

5. RESSLER, N., AND GOODWIN, J. F., Amer. J. Clin. Patho?. 38, 131 (1962).

6. GOA, J., Scund. J. Clin. Lab. Inoest. 5, 218 (1953).

7. ITZHAKI, R. F., AND GILL, D. M., Ana!. Biochem. 9, 401 ( 1964 ).

72 BiiRGI AND KAUFMANN

8. RICHTERICH, R., “Klinische Chemie: Theorie und Praxis,” 3rd ed., p. 499.

Karger, Basel, 1971. 9. BURGI, W., RICHTERICH, R., AND BRINER, M., Clin. Chim. Acta 15, 181 (1967).

10. ROBSON, R. M., GOLL, D. E., AND TEMPLE, M. J., Anal. Biachem. 24, 339 (1968).

11. SOKOLOWSKI, A., JUNG, K., AND EGGER, E., 2. Klin. Chem. Klin. Biochem. 10, 531 (1972).

12. PETRONICI, C., AND SAFINA, G., Chem. Abstl. 47, 11297-239, 247 (1953).

13. Roche Diagnostica Manual, Glucose Determination, Hoffmann-La Roche, Basel,

1972.

14. BENNET, TH. P., Nature (London) 213, 1131 (1967).

15. BECKMANN, R., AND PETITH, M., Med. Welt 21, 140 (1970). 16. PERLES, R., AND COLAS, M. C., C. R. Sot. Biol. 153, 395 (1959).

17. BAUMGARTEN, F., AND SAAR, M., Deut. Z. Nervenheilsk. 180, 125 (1960).

18. DAUGHADAY, W. H., AND LARNER, J., J. C&n. Inuest. 33, 326 (1956).

19. BA~AGLIA, F. C., MESCHIA, G., BLECHNER, J. N., AND BARRON, D. H., Quart. 1.

Exp. Physiol. 46,188 ( 1961).