Short Communication

A rapid MEKC method for the simultaneousdetermination of creatinine, 1- and3-methylhistidine in human urine

The present report describes the use of MEKC in the presence of 35 mM sodium

tetraborate, pH 9.0, containing 60 mM SDS for the complete separation and identifica-

tion of creatinine, 1-methylhistidine (1-MeH) and 3-MeH in human urine. Their

simultaneous quantification in urine of healthy controls and subjects submitted to

elective surgery to their lower limbs allowed to use 3-MeH as a reliable measure of

skeletal protein breakdown. Simplicity, sensitivity and low running costs are the main

advantages of this method that is suitable for the routine analysis in clinical laboratories

of a large number of samples per day.

Keywords:

Creatinine / MEKC / 3-Methylhistidine / Urine DOI 10.1002/elps.200800565

Methylation of histidine molecule in the 1- and 3-positions

of the imidazole ring, with formation of 1-methylhistidine

(1-MeH) and 3-MeH, respectively [1–4], is a well-known

post-translational modification of actin and myosin.

Although skeletal muscle accounts for 90% of the total-

body 3-MeH pool, significant amounts of this amino acid

are also produced by the hydrolysis of ingested muscle

protein. This prevents the potential use of its excretion rate

in urine (3-MeH released after protein degradation in fact

can neither be re-used for protein synthesis nor metabo-

lized) as an index of muscle protein breakdown, unless the

amount derived from diet is known [5–10]. Interestingly, the

determination of 1-MeH meets this demand. Although

1-MeH is common in other animals, it is not produced in

humans. Given that its urinary levels correlate well with

those of exogenous 3-MeH, the simultaneous determination

of these amino acids becomes a reliable tool to monitor

proteolysis of the skeletal muscle proteins [11].

Although a direct fluorimetric method has already been

described [12], measurement is usually performed either by

ion-exchange chromatography or high-performance liquid

chromatography [8]. More recently, Tuma et al. [13, 14]

applied CZE to enhance sensitivity and to avoid time-

consuming pre-treatment procedures. However, if this

approach was fast and sensitive, it did not provide excellent

resolution of the two analytes [13]. On the other hand, the

development of this procedure by applying contactless

conductivity detection, while improving separation, consid-

erably lowered sensitivity [14].

This report demonstrates that MEKC provides an

excellent resolution coupled to high sensitivity and fast

analysis times and hence may satisfy all of these demanding

criteria.

Analyses were performed on a P/ACE (Beckman

Coulter, Fullerton, USA) model 5000 instrument equipped

with a UV detection system. Untreated fused-silica capil-

laries Beckman (50 mm id� 57 cm total length, 50 cm to the

detector) were used. An aliquot of 35 mM sodium tetra-

borate, pH 9.0, containing 60 mM SDS was the BGE.

Separations were carried out at 251C by applying a voltage of

20 kV. Aliquots containing 5–8 nL of sample were intro-

duced in the capillary under a nitrogen pressure of 3.5 kPa

for 5 s and the analytes monitored at 214 nm. Between runs,

the capillary was washed for 2 min with 0.5 N NaOH solu-

tion. MS spectra were produced by using an ESI-Ion Trap

model LCQ Advantage (Thermo Finnigan, San Jose, CA,

USA) equipment.

The separation of standard analytes showed the typical

pattern recorded in profile ‘‘a’’ of Fig. 1A in which peaks

numbered 1 (migration time, tm: 5.2370.20 min),

2 (tm: 6.6570.25 min) and 3 (tm: 7.9470.22 min) represent

creatinine, 1- and 3-MeH, respectively. Calibration curves

(data not shown) of the three standard compounds were

produced by measuring (in triplicate) over a concentration

range between 10 and 0.005 mM. The correlation coefficient

(r2) was 0.9990 for creatinine, and 0.9980 and 0.9975 for

1- and 3-MeH, respectively. The LOD, defined as three times

the signal-to-noise ratio, was 10 mM for creatinine and 5 mM

for the other two analytes. The intra-day and inter-day

precision (RSD%) of the MEKC system was examined for

variations in tm and peak area/corrected peak area (A/cA in

which cA 5 A/tm) of the analytes’ peaks. The intra-day

Fabio Ferrari1

Marco Fumagalli1

Simona Viglio1

Roberto Aquilani2

Evasio Pasini3

Paolo Iadarola1

1Department of Biochemistry ‘‘A.Castellani’’, University of Pavia,Pavia, Italy

2Metabolic Service andNutritional Pathophysiology,Salvatore Maugeri Foundation,Montescano, Pavia, Italy

3Cardiology Division, SalvatoreMaugeri Foundation, Gussago,Brescia, Italy

Received September 2, 2008Revised November 25, 2008Accepted November 25, 2008

Abbreviation: MeH, methylhistidine

Correspondence: Professor Paolo Iadarola, Dipartimento diBiochimica ‘‘A. Castellani’’, Universita di Pavia, Via Taramelli3/B I-27100 Pavia, ItalyE-mail: piadarol@unipv.itFax: 139-0382-423108

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com

Electrophoresis 2009, 30, 654–656654

precision was calculated by performing (in triplicate) ten

injections (n 5 30) of a mixture containing the compounds

at a concentration of 0.5 mM each. The inter-day precision

was determined by performing (in triplicate) four injec-

tions/day of this mixture over four consecutive days, for

a total of 48 injections.

The values calculated for intra- and inter-day precision

of migration times (ranging between 1.14 and 1.27 and

between 1.20 and 1.34 RSD %, respectively) confirmed the

stability of the system. Furthermore, intra-day precision of

corrected area values ranged between 0.94 and 1.13 and

inter-day precision between 1.05 and 1.30 RSD %. Accuracy,

calculated as the difference in concentration between

measured and theoretical, provided recovery values for the

three analytes ranging between 96.7 and 99%. The robust-

ness of the method was assessed performing experiments in

which a series of operational parameters including BGE

(50 mM sodium phosphate containing 60 mM SDS), injec-

tion volume (2.5 or 10 mL) and inner diameter of the capil-

lary (75 or 100 mm id) were varied. Results (data not shown)

confirmed that resolution and recovery of analytes were

practically identical to those achieved using the conditions

described above. Thus, given the good reliability of this

procedure, we proceeded to apply it to real samples.

Figure 1. (A) Profiles obtained by sub-mitting to electrophoretic separation: amixture of standard analytes (trace a), arepresentative urine sample chosen atrandom from all those investigated(trace b) and the same sample as intrace b, spiked with a known amount(1 mM final concentration) of a mixturecontaining the three standards (trace c).Peaks 1–3 in all traces represent creati-nine, 1- and 3-MeH, respectively.Separations were performed by using35 mM sodium tetraborate pH 9.0containing 60 mM SDS as the BGE andby applying a voltage of 20 kV. (B) MSspectra obtained from peaks 1–3collected performing a series (n 5 150)of ‘‘preparative’’ runs. The typical signalat m/z 5 114.06 corresponds to the[M1H]1 ion of creatinine; the signals atm/z 5 170.02 and 170.06 correspond tothe [M1H]1 ions of 1- and 3-MeH,respectively.

Electrophoresis 2009, 30, 654–656 CE and CEC 655

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com

A total of 44 urine samples, 18 of which were from

healthy volunteers (controls) and 26 from patients who had

been submitted for elective surgery to their lower limbs (hip

and knee arthroprotesis, 12 and 14 patients, respectively),

were analyzed in triplicate without being submitted for any

pre-treatment procedure. Given the identity of profiles of

controls and patients, electropherogram shown in trace ‘‘b’’

of Fig. 1A may be considered representative of all available.

This pattern contained, in addition to others, three peaks

(numbered 1–3) whose migration times (tm: 5.2270.22,

6.6470.26 and 7.9570.22 min) corresponded exactly to

those of standard compounds (see values reported above).

To confirm their identity, a number (n 5 25) of urine

samples were spiked with a known amount (5 mL of a

10 mM solution; final concentration in the sample 1 mM

each) of standards and re-injected. As shown in trace ‘‘c’’ of

Fig. 1, which shows a typical pattern of spiked samples, the

perfect overlap of reference compounds peaks with those of

endogenous analytes promoted in co-injected samples a

proportional increase in the height of peaks numbered 1–3.

However, since the criterion of spiking samples with the

appropriate standard and monitoring their peak co-migra-

tion does not exclude the possibility that other components

of urine may co-elute with the spiked standards, MS

was used to assign unambiguously the peaks to the

above-mentioned analytes. This analysis was carried out

(i) performing a series (n 5 150) of ‘‘preparative’’ runs,

(ii) collecting the material hypothetically corresponding to

these analytes and (iii) submitting it to MS analysis. As

shown in Fig. 1B, the typical MS signals (m/z 5 114.06

corresponded to the [M1H]1 ion of creatinine;

m/z 5 170.02 and 170.06 corresponded to the [M1H]1 ions

of 1- and 3-MeH, respectively) not only confirmed the identity

of analytes but also assessed the absence of contaminants. As

deduced by our calculations, the amount of 3-MeH excreted

in urine by the two groups was apparently similar, the mean

values being 0.1170.05 and 0.1870.04 mm/mL for controls

and patients, respectively. However, the simultaneous deter-

mination of 1-MeH (corresponding to the amount of

exogenous 3-MeH) and of creatinine allowed to estimate that

the percent of muscle protein degraded each day by the group

of patients was three times higher than that of controls. The

application of the expression previously reported by Ballard

and Tomas [8] produced a mean value of muscular degra-

dation (%) of 2.20 for the subjects of the former group against

a value of 0.8 for those of the latter.

Although not surprising, given the important metabolic

alterations experienced by patients, this finding was of great

interest since it confirmed the reliability of the method in

revealing significant differences in the amount of muscle

degraded by individuals under different clinical conditions.

In conclusion this procedure seems to be a viable

alternative to existing methods. Given its simplicity, sensi-

tivity and low running costs, it is suitable for routine

analysis in clinical practice of a large number of urine

samples.

The authors thank Mr. Giuseppe Zucchinali (BeckmanCoulter, Milan, Italy) for his friendly co-operation with the CEequipments and Dr. Robert Coates (Centro Linguistico,Universita Bocconi, Milan, Italy) for his linguistic corrections.

The authors have declared no conflict of interest.

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