Electrophoresis 1988, 9, 839-841 Determination of serum oxalate by multicolumn isotachophoresis 839

Short communications

Vladislav Dolnik Mirko Deml Petr BoEek

Institute of Analytical Chemistry, Czechoslovak Academy of Sciences, Brno

Determination of oxalate in human serum by multicolumn isotachophoresis

The practical usefulness of multicolumn isotachophoresis was demonstrated by the determination of oxalate levels in human serum. 10 mmol/L HCI and 100 mmol/L Na,HP04 served as the leading and terminating electrolyte, respectively. For the stabilization of the isotachophoretic zones in large cross section channels a suspen- sion of Bio-Gel P-300 in the leading electrolyte was used. For the analysis ca. 1 mL of fresh serum was required and 1.2 mL of 1 : 1 mixture of serum and the suspension of Bio-Gel P-300 in deionized water was applied as sample. The time of analysis ranged between 45-49 min. The detection limit of analysis was determined to be 50 nmol/L. Reproducibility of analysis of 1 pmol/L oxalate in water standard was found to be 2.6 % (M = 6).

Capillary isotachophoresis has already proved to be useful for analyzing ionogenic components in body fluids (for a survey see monographs [ 1,21 or recent reviews [3-61). Advanced in- strumentation of the technique, especially the column-cou- pling system [71, proved to be suitable for practical analytical problems with sample components ranging within mol/L (CJ: [51). The recently published multicolumn system [5, 81 has shown its potential to analyze components with direct sample injection at the level of 10-6mol/L, thus reaching the concentration level of significant metabolites in body fluids. The practical usefulness of multicolumn isotachopho- resis is demonstrated in this paper by the determination of oxalate levels in human serum.

To determine oxalate levels in blood, plasma or serum, several methods were used and various physiological ranges were observed: radioenzymatic methods: 2.26 & 1.67 pmol/L [91, 3.4 k 0.7 pmol/L [ 101,9.2 k 2.6 pmol/L [ 1 1 I , 4.0 pmol/L 1 121, 13.0 f5.1 pmol/L[13l,gaschromatography: 2.8 ? 1.1 pmol/ L [141,4.9 k0.8 pmol/L [151,liquidchromatography:32.3 & 9.9 pmol/L [161, 20.0 ? 5.7 pmol/L (calculated from the published results) [ 171. If the oxalogenesis in vitro(i. e. a spon- taneous generation of oxalate after blood samp1ing)is not con- trolled, the oxalate concentrations found are consistently higher [9, 10, 12-141. Higher levels of plasma oxalate found by liquid chromatography [ 16,171 can be explained by this ef- fect. Several procedures were recommended to suppress the spontaneous generation of oxalate in blood samples: (i) blood sampling in presence of a mixture of enzyme inhibitors [9l (however, the usefulness of this method was not confirmed by other researchers [lo, 12-14]), (ii) acidification of plasma to pH < 3.8 [ 131, (iii) sample processing not later than 3 h after blood collection [14].

Bio-Gel P-300 was obtained from Bio-Rad Laboratories (Vi- enna, Austria). All other chemicals were supplied by Lach- ema (Brno, Czechoslovakia). The isotachophoretic analysis was performed in the equipment for multicolumn isotacho- phoresis as described elsewhere [8]. The migration unit con- sists of a separation channel (cross section 20 x 1.3 mm), two tapered channels, one reestablishing channel (cross section 1 x 0.5 mm) and one detection channel (diameter 0.2 mm). The

Correspondence: Dr. Vladislav Dolnik, Institute of Analytical Chemistry. Czechoslovak Academy of Sciences, Leninova 82, CS-611 42 Brno, Czechoslovakia

sample valve has a sampling volume of 1.2 mL and is con- nected to the separation channel. Thelarge cross section chan- nels are insulated from the cooling block by Bytac VF 8 1 foil (Chemoplast, Norton, Wayne, USA). The detection capillary and the large cross-section channels together with appropriate buffer volumes are filled with the solution ofleading electrolyte and with the suspension of leading electrolyte in granulated gel, respectively. A power supply, VNZ 22 (Vyvojove dilny CSAV, Prague, Czechoslovakia) with optionally stabilized voltage, current and power (0-2 kV, 0-200 pA, 0- 100 W), was used for the separation step. In the following steps a laboratory-made power supply with stabilized d.c. current up to 550 pA (max. voltage 16 kV) or with stabilized voltage ca. 2 kV (maximum current ca. 5 mA) was used [181. The iso- tachophoretic zones were detected by a potential gradient detector [ 181.

The composition of the leading electrolyte was 10 mmol/L HCl in a suspension containing Bio-Gel P-300 and 10 mmol/L HCl with addition of 0.3 % polyethylene glycol 6000 in wide cross section channels and in the detection capillary, respec- tively. As terminating electrolyte 100 mmol/L Na,HPO, in a suspension of Bio-Gel P-300 was used. Blood samples were kept in a refrigerator for 30 min to clot and were then centri- fuged. One mL of serum was mixed with an equal volume of a Bio-Gel P-300 suspension in deionized water. The resultant suspension was introduced into the sampling valve with a 1.2 mL volume. The time interval between blood collection and start of analysis was less than 45 min.

The blank run has shown two impurities in the isotachophero- gram (see Fig. 1). The first one having higher effective mobility than oxalate seemed to have its origin in the leading electrolyte and was not found in the samples containing a higher con- centration of chloride. The second impurity had approximate- ly the same effective mobility as oxalate. The usual purifica- tion procedures applied to the chemicals used for the prepara- tion of electrolyte solutions were not successful and the im- purity was always present in the isotachopherogram of the blank run. Therefore; the content of this impurity has a great influence on the detection limit of oxalate. The apparent con- centration of impurity having the effective mobility equal to that of oxalate was found to be equivalent to 0.18 pmol/L in blanc runs with a standard deviation of 16.7 nmol/L in 6 runs. Hence the detection limit (3 s ) was determined to be 50 nmol/L.

0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1988 01 73-0835/88/12 12-0839 $02.50/0

840 V. Doliiik, M. Deml and P. BoEek Electrophoresis 1988.9, 839-841

t I

I t 28 28.imin

Figure 1 . Isotachopherogram of blank.

For quantitative analysis the calibration curve coverzd the range 0-10 ymol/L, obtained by analyzing a series of stand- ard solutions (see Fig. 2). The calibration line (see Fig. 3) was linear in this range and can be expressed by the equation

Qoxalate[mCI =0.116 + 1.129 Coxalate[~mol/LI

where Qoxalate is the zone passage charge of the oxalate. When analyzing blood serum the total analysis time ranged from 45-49 min. The separation step required 27 min at aconstant driving current of 30 mA, the skidding step (i. e. the transfer of oxalate from the end of the separation channel to the near end of the detection capillary) took ca. 4.5 min at a constant voltage of 550 V and thedetection step (i. e. the final analytical run) ca. 14 min at a constant driving current of 100 PA. In the isotachopherogram of blood serum (Fig. 4), the zones of oxalate and of a non-identified component of blood are well separated. For an estimation of the physiological range of oxalate in serum, seraof 3 apparently healthy malevolunteers, aged 30-50, were analyzed and 5.5, 5.4 and 5.7 pmol/L of oxalate were found.

In the described method phosphate served as the terminator so that other organic acids present in blood, either connected with metabolism of oxalate (glycolate. glyoxalate and ascor- bate) or those of high clinical importance (lactate, acetoace- tate, 3-hydroxybutyrate, etc.) could not be determined. At the pH of the leading electrolyte, pH 2.0, all these acids mi-

t i

i-' L 28 28.5min t

Figure 2. Isotachopherogram of 1 pmol/L oxalate. Analysis of 1.2 mL of standard in a Bio-Gel P-300 suspension.

Figure 3. Zone passage charge of oxalate vs. oxalate concentration in sample as the calibration line.

r

u . 1rnC

48 48krnin t

Figure 4 . Isotachopherogram of serum of a healthy individual. Analysis of 1.2 mLofserumdiIuted 1:1 by thesuspensionofBio-GelP-3OOin deionized water.

Table 1. Dependence of serum oxalate level on the age of sample

Interval from blood collection h

Oxalate level Nmol/L

1 2 3

5.7 6.8 7 .5

grate isotachophoretically behind phosphate. The content of phosphate in serum is rather high (ca. mol/L) and it can- not be removed from the system in the meantime by migra- tion to the auxiliary electrode. This is the reason why the si- multaneous analysis of oxalate and other blood acids is not feasible - since the time of the whole analysis would exceed several hours. It was serum and not plasma that was used as sample, because the addition of anticoagulants (heparin, fluoride, citrate) to blood led to a substantial elevation of the measured concentrations of oxalate. Oxalogenesis in vitro [ 9, 10, 12- 141 was confirmed by elevated oxalate concentrations with an increasing time interval between blood collection and start of the analysis (see Table 1). However, our results indicate that a 3-h storage of sample is too long, which is in contrast to ref. [14]. Due to the oxalogenesis in vitro the reproducibility of serum analysis is hard to evaluate by

Electrophoresis 1988, 9, 84 1-844 Cross reaction of plant glycoproteins 84 1

repeated analyses of the same serum sample. For that reason, reproducibility of the analysis of a water standard was only tested and, using a 1 pmol/L standard solution, the relative standard deviation was found to be 2.6 % (n = 6).

161 Dolnik, V. and BoEek, P., Chem. List? 1986,80,28-5 1. 171 Everaerts, F. M., Verheggen, T. P. E. M. and Mikkers, F. E. P.,

[ S l Dolnik, V., Deml, M. and BoEek, P., J. Chromatogr. 1985, 320, J. Chromatogr. 1979, 169, 21-38.

89-97.

Received April 29, 1988

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Anne-Catherine Laine Significant immunological cross-reactivity of plant Lok Faye gl ycoproteins CNRS, UA 203, Mont Saint Aignan

Plant glycoproteins generally cross-react because of the presence of identical or related complex glycans which are highly immunogenic. The use of mild periodate oxidation of glycans after glycoprotein transfer from sodium dodecyl sulfate-poly- acrylamide gel electrophoresis gels to nitrocellulose membranes prior to immunode- tection is a way of identifying the carbohydrate antigenic determinants of a glyco- protein as the basis for antigenic cross-reaction. Periodate oxidation can distinguish between antibodies directed against carbohydrate and against peptide antigenic determinants, the latter being unaffected by oxidation. Immunoblotting performed after periodate treatment allows the detection of common protein epitopes.

Plant and animal cells contain glycoproteins with N-linked glycans of the polymannose or of the complex type. Polyman-

' nose glycans have identical structures of the type Mans-9 (G1cNAc)z in plants and animals. However, the complex glycans of plant glycoproteins differ from those described in mammals by the absence of sialic acid and by the presence of a fucoseresidue a, I-+3-linked to the innermost GlcNAc andofa xylose residue P, lb2-linked to the P-linked mannose of the core. It has long been assumed that the predominant specific- ities of antibodies raised against plant glycoproteins are directed toward the protein moiety. However, recent results clearly show that a polyclonal antiserum raised in rabbit against a plant glycoprotein contains mostly, if not exclusive-

Correspondence: Dr. LoYc Faye, Centre National de la Recherche Scien- tifique, UA 203,Universitkde Rouen, Faculte des Sciences, F-76130 Mont Saint Aignan, France

Abbreviations: ConA, concanavalin A; GlcNAc, N-acetylglucosamine; PHA, phytohemagglutinin

ly, antibodies specific for the complex glycan of this glycopro- tein [ l l . The complex glycans of plant glycoproteins have, most commonly, the composition Man,XylFuc(GlcNAC)2. In this paper we show that the widespread occurrence of highly immunogenic carbohydrate moieties and anti-glycan antibodies in antisera raised against plant glycoproteins may be the basis of antigenic cross-reactions among glycoproteins with distinct polypeptide structures.

To investigate plant glycan immunogenicity we have used polyclonal antisera raised in rabbits against different plant glycoproteins according to various immunization schedules [2,31. The specificity ofthese immune sera was studied by im- munoblotting. Proteins from different extracts: French bean seeds, jackbean seeds, radish seedlings and cell wall from suspension culture carrot cells were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) according to Laemmli [91 and transferred to a nitro- cellulose membrane. Visualization is performedusing aperox-

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