Determination of L-Hydroxyproline in Meat Protein by Quantitative Carbon- 13 Fourier Transform Nuclear Magnetic Resonance Spectrometry M. L. Jozefowicz, I. K. O'Nelll," and H. J. Prosser Laboratory of the Government Chemist, Cornwail House, Stamford Street, London SE1 9NQ, U.K.
13C FTNMR spectroscopy is found to provide an efflcient assay technique for connective tissue in meat, by analysis of Lhydroxyproline in defatted, hydrolyzed meat. The sensitivity of 13C FTNMR spectroscopy is increased by rapid pulsing of highly concentrated vlscous solutions, and quantttative analysis is achieved by comparlng the peak height of C-4 in Lhydroxyproline with that d an internal standard. The technique has dlstinct advantages over exlsting colorimetric and amino-acld analysis prwedures. Most meats and processed meats contain 1 to 2% by weight Lhydroxyproiineof the dry, defatted meat; a quantltative analysis taking 29 mln is found to have a standard deviation of 0.16% at a level of 3.0% w/w Lhydroxyproline using a commercially available spectrometer with 10mm 0.d. NMR tubes and a crystal fitter. Survey spectra of Increased sensitivity are obtalned by shorter or longer observatlon as desired.
Connective tissue imparts strength t o tendon, skin, and muscle tissue but increases the toughness of meat as a consequence (I). In order to utilize a wider range of carcass meats, modern meat processing techniques reduce toughness (2) but thereby necessitate a method to detect connective tissue both as a labeling safeguard and as a guide to nutritional value. Collagen, the principal connective protein, is the only protein to contain significant levels of L-hydroxyproline, I, OH
\ L
H
I which is a non-essential amino acid (3). As the two present methods of L-hydroxyproline assay have distinct disadvantages in specificity or sensitivity, 13C Fourier transform nuclear magnetic resonance (FTNMR) spectroscopy was applied to this problem after some initial encouraging results ( 4 ) . 13CFTNMR spectroscopy (5) has unsurpassed specificity for mixtures of polar organic substances and has been used to study the properties of purified proteins (6). However, it suffers from lack of sensitivity and inherent nonquantitation (7) unless precautions are taken. One purpose of this work was to obtain routine quantitation using this highly specific technique, thereby providing an alternative to labor-intensive separative procedures. The immediate purpose was to obtain a routine L-hydroxyproline assay at least as efficient as existing methods.
EXPERIMENTAL Materials. L-Hydroxyproline, I (puriss, Koch Light) and fi-hydroxy-a-methylphenethylammoniumchloride (phenylpropanolamine hydrochloride), the standard I1 (99+ %, Aldrich) were used as received. Collagen (acid insoluble ex bovine Achilles 1140
ANALYTICAL CHEMISTRY, VOL. 49, NO. 8, JULY 1977
Table I. Spectroscopic Conditions Used for Quantitative W-FTNMR of L-Hydroxyproline Frequency 13Cobservation 25.03 MHz Observation frequency range 6.25 KHz Frequency filter range Frequency 'H irradiation 99.5 MHz 2.5 KHz ' H noise modulation range Frequency 2Hlock 15.0 MHz Pulse width 15 ps ( 7 5" tip angle) 2048 Number of pulses Data acquisition time 650 ms Repetition time 850 ms 260 p s Acquisition trigger delay FID data points 8191 FID apodization Exponential -5 Sample temperature 80 "C Total observation time 29 min tendon and acid soluble ex calf skin) and poly-L-hydroxyproline were all from Sigma London Chemical Co and used as received. Techniques. Meat samples were freeze dried, ground in a Moulinex coffret grinder, then defatted under reflux with chloroform/methanol(1:l v/v). The dry product was ground again and a 2-g portion hydrolyzed in 40 mL 6 N hydrochloric acid at 120 "C for 24 h in a glass/Teflon pressure vessel. After partial concentration in vacuo at 50 "C, a weighed portion of standard (223 mg for 2 g dry sample) was dissolved. In establishing calibration graphs, poly-L-hydroxyproline was added before hydrolysis, or L-hydroxyproline added at the same time as the standard, All results are expressed as weight percent of the dried defatted meat samples. A viscometric method was used to monitor completion of the concentration process so as to yield a black gum of viscosity approximately 7000 cSt at 23 "C. Viscosity was measured by timing the flow up an evacuated standard capillary, calibration being effected with a range of polydimethylsiloxane fluids. I3C-FTNMRspectra were obtained of samples in 10-mm 0.d. tubes with a JEOL PFT-100 spectrometer fitted with a 5-KHz crystal filter, 24K computer memory and a software controlled pulser. A deuterium field-frequency lock was made with 1 N DCl/D20 in a sealed 2-mm 0.d. coaxial capillary tube. The conditions of sample examination are listed in Table I, unless otherwise indicated in the text. Spin-lattice relaxation times were measured by the 18O0-~-9OPtechnique (8). Samples with exceptionally low L-hydroxyprolineabundance were examined under slightly different conditions to overcome the limited dynamic range of the computer memory. A frequency range of 2.5 KHz and a frequency filter range of 800 Hz were used. The irradiation frequency was set 200 Hz higher than the Lhydroxyproline C-4 resonance frequency and data were accumulated into 4095 data points. L-Hydroxyproline was also determined (a) with a Technicon NC1 amino acid analyzer fitted with a polysulfonated polystyrene column (8% cross-linked with divinylbenzene) and ninhydrin colorimetric detection, (b) by a modification of the Stegemann colorimetric method (9) using a Technicon AutoAnalyzer. 13C abundance levels were determined by Stable Isotope Analytical Services, London. Relative to the international Petroleum Data Base COz standard, collagen and the standard I1
i
HYP
210
150
100
50
b,l 6w
Figure 1. 25.03 MHz %-FTMNR spectra of collagen (a) In 6 M guanidinium chloride at 100 OC (30-min observation), (b) acid hydrolysis product in 1 N HCI at 50 OC (10-min observation, C-4 resonance of L-hydroxyproline arrowed). 10-ps pulses were applied with 1.O-s repetition time to both samples
C
I
have a I3C deficit of 13.8 and 1.3 parts per thousand, respectively, thereby signifying a 1.25% I3C abundance difference. ZOO
RESULTS AND DISCUSSION Detection of L-Hydroxyproline in Meat. The 13CFTNMR spectrum of acid-insoluble collagen (Figure la) consisted of broad lines of low sensitivity, typical of the spectrum for a relatively rigid polypeptide (10). By contrast, acid-hydrolyzed collagen had a 13C-FTNMRspectrum with sharp lines of much greater sensitivity (Figure lb). The signal for C-4 of L-hydroxyproline fortuitously appeared at the extreme downfield end of the aliphatic carbon region (arrowed in Figure l b ) thus aiding detection. The reduction of linewidth arises from both the greater mobility of the uncombined amino acids and from elimination of the multiplicity of chemical shift increments arising from secondary and tertiary polypeptide structure. The increased signal/noise ratio can be attributed to the reduced line-width, but also to reduced spin-lattice relaxation time, T1. Thus collagen in the hydrolyzed state gave a much superior qualitative indication for L-hydroxyproline than the polypeptide itself. Samples of meat showed similar 13Cspectral characteristics. Thus the 13C-FTNMR spectrum of whole meat (Figure 2a, chicken) was dominated by the relatively mobile fat and no sign of any protein could be found in its natural state. Defatted, denatured chicken protein gave a better defined I3C-FTNMR spectrum Figure 2b) than that of denatured collagen (Figure la), but hydrolysis was necessary to yield a spectrum of the desired resolution and sensitivity (Figure 2c). A small peak appeared in the position expected for the C-4 resonance of L-hydroxyproline. Other meat samples of known high collagen content exhibited a more intense peak in the same position. As collagen gave an inferior 13C-FTNMR spectrum compared with other meat proteins in the polypeptide state (compare Figures l a and 2b), it was decided to examine meat only after acid hydrolysis. Existing methods of L-hydroxyproline assay also require prior acid hydrolysis of meat which has been freeze-dried and defatted. The same hydrolysis procedure was adopted except that it was scaled up to provide enough hydrolysate for convenient I3C-FTNMR detection. Sensitivity. Saturation generally limits the sensitivity achieved for a signal in 13C-FTNMRspectroscopy (5). If the spin-lattice relaxation time (TI) can be reduced, more irradiating pulses and/or pulses of greater intensity can be applied to the sample without saturation in the same time. Therefore the Tl for the C-4 resonance of L-hydroxyprolinewas studied as a function of sample viscosity and temperature.
150
100
50
0
wrn
Figure 2. 25.03MHz 13C-FTNMR spectra of chicken meat. (a)Excised portion of breast muscle at 25 OC (16-h observation),(b) defatted dry product in 6 M guanidinlum chloride at 100 OC (30-min observation), both spectra obtained with 10-ps pulses applied with 1.O-s repetition time. (c) Defatted hydrolyzed product in 1 N HCI at 80 'C (30-min
accumulation, conditions in Table I)
Preliminary experimentswith the meat hydrolysates showed that these solutions could be highly concentrated to high viscosity without apparent precipitation of the amino acids. Furthermore, 13C-FTNMRspectra of useful resolution were obtained at elevated temperatures from viscous samples that gave no useful spectrum in ambient conditions. Increased hydrolysate concentration was beneficial for sensitivity in two ways; the T1 was greatly reduced by high viscosity and the sample concentration increased. However, in very concentrated solutions (>80% w/w) the spectral lines were unacceptably broad. Thus a balance between the related effects of concentration, viscosity, and temperature was required, and established, for optimum sensitivity in a given time. Information theory (10) sets a minimum data acquisition time after each pulse for any specific number of data points per unit of spectral width; for adequate peak representation, an acquisition time of 650 ms was found necessary. For this reason, solutions of protein hydrolysates were concentrated to approximately 70% w/w giving a T1of about 100 ms for L-hydroxyproline C-4 at 80 "C. The sensitivity for usual samples containing 1 to 2% L-hydroxyproline by weight of the dry defatted meat was found adequate after half-hour accumulations (Figure 2c) under these conditions. Choice of Internal Standard. Quantitative NMR spectroscopy (I1 ) has been extensively performed with continuous wave spectrometers observing 'H, 31P,or "F, usually using an internal standard within the sample and assuming that peak area is proportional to the concentration of the nucleus of interest. The pulse-Fourier transform technique for 'H-decoupled 13C observation, as used here, has a number of inherent problems that preclude straightforward quantitation of this kind (7). Firstly, differences in TI and nuclear Overhauser enhancement (nOe) must be expected for any two 13C nuclei. With a T1 for the C-4 resonance of Lhydroxyprolinereduced to about 100 ms, an internal standard with similar TI would eliminate selective saturation (12)since the established minimum pulse repetition time was 650 ms. Secondly, unequal pulse excitation or detection arises from the frequency separation of two signals being compared. The ANALYTICAL CHEMISTRY, VOL. 49, NO. 8, JULY 1977
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within the sample. The L-hydroxyproline and standard peaks were assessed both by integration and by peak height. Comparison of relative peak heights was found better providing care was taken in setting the baseline for measurement. Previous workers have found FTNMR integration software to be lacking (13); others have prepared new software for a hybrid method using the top nine digital values for each peak
a
I
(14).
I 200
150
100
50
0 PPrn
b
-6
6 ppm-
Flgure 3. 25.03 MHz I3C-FTMNR spectrum of defatted, hydrolyzed turkey breast with 4 % w/w L-hydroxyproline and 5 % w/w standard I1 at 80 OC (30-min observation, conditions in Table I); (a) a full spectrum, (b) expanslon
use of a crystal filter aggravated this situation and dictated that a suitable internal standard should have minimal separation in the I3C-FTNMR spectrum from the C-4 resonance of L-hydroxyproline. Thirdly, the standard had also to be soluble and stable in the hot, highly polar, acid conditions used in sample preparation and I3C data acquisition. P-Hydroxy-a-methylphenethylammoniumchloride, 11, was found suitable with the P-carbon resonance a t 2.9 ppm downfield from the L-hydroxyproline C-4 resonance, and with TIof approximately 80 ms. This standard was found to be stable in the experimental conditions and only after 10 h a t 80 OC was significant decomposition observed.
?H N H 2 * HCI
I1 Two small effects can also be expected in comparing one 13Cresonance with another. Minor variations in 13Cnatural abundance are known to occur and so the 13Cabundances were measured for collagen and the standard, 11. A difference of 1.25 f 0.03% in 13Clevels was found but is not significant in the present work. Signal intensity is also lost by coupling with adjacent I3C nuclei (1.1%loss per attached carbon) and so it is better to have a standard with an identical substitution pattern, as here (two carbons, one hydrogen, one oxygen). Calibration. Turkey breast was selected as a meat with a low natural level of L-hydroxyproline for preparation of a calibration graph. Defatted, freeze-dried turkey breast was f i e l y ground so as to homogenize any fibrous connective tissue and portions were hydrolyzed in the standard conditions. After partially concentrating the hydrolysate, the internal standard and L-hydroxyproline were dissolved and then the sample was concentrated (to an approximate viscosity of 7000 cSt a t 23 OC) prior to pouring into the NMR tube. This procedure was found reproducible and ensured homogeneity of the sample at each stage from protein through to NMR spectroscopy. Small variations in sample viscosity had no deleterious effect on the NMR measurements. 13C-FTNMR spectra (see Figure 3) were performed with locking on a coaxial capillary containing 1 N DC1/D20; this gave much better resolution than by locking on to deuterium 1142
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The results showed that the peak height ratio increased linearly with added hydroxyproline, thereby indicating that neither L-hydroxyproline or the standard precipitated or decomposed in the sampling process. The negative intercept corresponding to 0.22 % w/w L-hydroxyproline was consistent with the low (
