Sep 6, 1983 - (102.611) characteristic resonances are those of the methyl group of alanine ..... We are very grateful to Rio Tinto Zinc Services,the Arthritis and.
CLIN. CHEM. 30/3, 426-432
(1984)
Use of High-Resolution Proton Nuclear Magnetic Resonance Spectroscopy for Rapid Multi-Component Analysis of Urine John R. Bales,1 Denise P. Higham,’ Ian Howe,’ Jeremy K. Nicholson,2and Peter J. Sadler”3 Numerous lOWMr metabolites-including creatinine, Citrate, hippurate, glucose, ketone bodies, and various amino acids-have been identified in400- and 500-MHz proton nucle-
Materials and Methods
ar magnetic resonance (1H NMR) spectra of intact human urine. The presence of many of these was related to the specific condition of the donors: humans in different physiological states (resting, fasting, or post-exercise) and pathological conditions (e.g., diabetes mellitus, cadmium-induced renal dysfunction). We have also monitored the metabolism of simple nonendogenous compounds (methanol and ethanol) and of acetaminophen. The pH-dependencies of the NMR chemical shifts of some urine components are reported. Our studies show that high-resolution 1H NMR spectroscopy provides a fast, simple method for “fingerprint” identification of urinary compounds. In some cases, analytes can be quantified by standard additions or by comparing integrated peak areas for the metabolites with those for creatinine. Determinations of creatinine by 1H NMR spectroscopy compared well with those by an independent chemical assay based on the Jaff#{233} reaction.
Specimens of the first morning urine were collected from normal healthy men and women. Urine was also collected from a healthy male volunteer before and after vigorous
Addftlonal Keyphrases: variation, source of diabetes toxicology methanol ethanol acetaminophen creatinine glucose ketone bodies amino acids monitor-
-
-
ing therapy with insulin
The development of high-field nuclear magnetic resonance (NMR) spectrometers with increased sensitivity, resolution, and dynamic range has made possible the rapid study of complex mixtures of compounds with little or no pretreatment. Proton NMR spectroscopycan provide details on the concentration and mobility of many low-molecularmass (Mr) metabolites in tissue (1) and in the blood and plasma of several animal species,including humans (2-5). It can also be applied to the study of metabolite concentrations in plasma in disease states such as diabetes mellitus, and preliminary observations suggest that urinary metabolites might also be usefully studied (6-8). In this paper we report the use of high-resolution ‘H NMR spectroscopy for the detailed analysis of urine components, mcluding lactate, 3-r-hydroxybutyrate acetoacetate, acetone, creatinine, glucose, citrate, and various amino acids
and their derivatives. The technique is rapid, requires only a small sample, and involves no separation or derivatization steps. The analytes studied can be detected down to about 100 p.mol/L. A major advantage is that the ‘H NMR signals for creatinine provide an internal standard. The integrated peak areas for many metabolites can be compared directly with those for creatinine and the concentrations expressed in terms of creatinine. 1
Department
of Chemistry,
Birkbeck
College,
London,Malet St., LondonWC1E 7HX, U.K.
University
of
2Toxicology Unit, Department of Pharmacology, School of Pharmacy, Brunswick Square, London WC1N lAX, U.K. 3To whom correspondenceshouldbe addressed. Received September 6, 1983;accepted December 12,1983. 426
CLINICAL CHEMISTRY, Vol.30,No.3, 1984
Subjects
exercise (a 1-h run). Another healthy 26-year-old male volunteer fasted for 48 h, during which he consumed only water (ad lib.) and supplied several urine specimens. Additional urine specimens, supplied by St. Thomas’s Hospital Medical School, London, were obtained from an insulin-dependent diabetic patient before and up to 12 h after voluntary insulin deprivation under controlled experimental conditions. For two days, five male Swiss A2G mice, five weeks old, were given ad jib, food and drinking water containing 40 mL of Analar-grade methanol per liter. The mice were then killed, and we sampled urine from their bladders by syringe and arterial blood directly from the heart by cardiac puncture. The plasma was separated by centrifugation. A clinically normal man ingested two 500-mg tablets of acetaminophen (N-acetyl-p-aminophenol) after an overnight fast. Urine samples were collected 5 mm before and 60, 98, and 124 mm after ingestion. Finally, we studied urine from a man known to have kidney damage after many years of industrial exposure to cadmium.
Samples The urine was collected in glass or polypropylene tubes and either used without delay or promptly frozen in liquid nitrogen and stored at -30 #{176}C until use. Samples (0.3-0.5 mL) containing 100 mL of added 21120 per liter as an internal frequency-field lock were placed into 5-mm (o.d.) glass NMR tubes. Alternatively, we lyophilized and redissOlved fresh urine samples in 2H20 at a 10-fold greater concentration before acquiring the spectra. Standards of various compounds (from Sigma Chemical Co., Poole, U.K.) were added to urine samples in 2H20 solution to give a final concentration of about 1 mmolJL. We also prepared in 21120 a solution of creatinine, citrate, glycine, and hippurate (about 100 mmol each per liter). We adjusted the PH” in steps from 4 to 8 with NaO2H and 2HC1 solutions (about 10 moltL each), and recorded the ‘H NMR spectra at various intervals. -
NMR Spectroscopy We used Bruker AM500, WH400, and JEOL FX200 spectrometers (operating in quadrature mode at 500, 400, and 200 MHz, respectively), with a Bruker Aspect 2000 data system. All spectra were recorded at ambient probe temperature (25 ± 1 #{176}C). The detection of weak NMR signals, such as those from urinary metabolites, in the presence of very large signals, pH”, the pH-meter
reading
in 2H20.
such as that from the water protons (110 mol/L), can pose a serious dynamic range problem. This arisesfrom limitations in the wordlengths of both the analog-to-digital converter (ADC) and the computer memory, which together influence the time required to achieve the maximum signal-to-noise ratio (9). The WH400 and AM500 spectrometers have 12 and 16 bit ADC’s, respectively; the latter provides a very large dynamic range (2 greater than that of the WH400), thus allowing the study of metabolites in urine at millimolar concentrations without the need to suppress the water signal. However, we obtained spectra with superior signalto-noiseratios by suppressing the large water signal by proton irradiation at the water frequency, either continuously or by presaturation by gated decoupling during the delay between pulses. These allowed us to increase the receiver gain for enhanced sensitivity. Alternatively, we applied a DANTE pulse train (10), consisting of 16000 pulses, each of 1-us, at 300-is intervals, at the water resonant frequency; this selectively saturates the water resonance but leaves the decoupler channel of the spectrometer free for
To test precision and reproducibility, we recorded the ‘H NMR spectra of five identical urine samples under the same experimental conditions: 64 accumulations at 24#{176}C, a 60 pulse, a 5.7-s interval between pulses, 16 384 data points, and continuous proton irradiation at the water frequency. After adding standard amounts of valine (50 L of a 207 mmolJL solution in 21120) to each sample, we re-recorded the spectra under the same conditions. Creatinine concentrations were determined by comparing computer-integrated peak areas for the CH3 resonance of creatinine with those of the two valine CH3 resonances. There was also a reasonable correspondence between the ratios of peak areas and peak heights when the digital resolution was high enough, i.e., when there were sufficient points per peak. Creatinine was independently determined in the same samples by the standard Jaffe reaction (13).
selective irradiation.
Figure 1 shows a typical spectrum for urine from normal human subjects.A reasonable signal-to-noise ratio was usually achieved after as few as 48 accumulations, depend-
Results and Discussion Normal Urine Samples
For some spectra we used a Hahn spin-echo sequence (11) (90 #{176}-ito 180 #{176}-T-collect), with additional water irradiation. This can aid interpretation of spectra because resonances are phase-modulated according to their multiplicity and coupling constants (12); e.g.,with r = 60 ms, singlets and triplets are upright, whereas doublets appear inverted if J = 8 Hz (r = 1/2J).
[I
ing on the dilution of the urine sample. Therefore, even allowing for a long relaxation delay, we could obtain a spectrum within 5 mm. We were able to identify the source of many resonances by making standard additions of candidate compounds and by considering their chemical shifts
T9
H1SIC4H) His ( C2 H)
Urea
Fm
9
7
8
6
S/ppm
Hp
Gly
Cr
Cr +Cn
U1
Ci
Ala
Acac Ha
Lac
8u
I] DEl
4
I
I
3
2
1
S/ppm
Fig. 1.400 MHz 1H NMR spectrum ofnormalhuman urine containing 100 mL 2H20 per liter Upper:aromatic region;Lower, aliphaticregion.These spectra are the result of 232 accumulations at 24 C, usinga 60 pulse,9.7-sintervalbetweenpulses, and 16384 data points.The waterresonancewas suppressedby continuousirradiation.An exponentialfunctioncorrespondingtoa 1-Hzlinebroadeningwas applied.Abbreviations for peak assignments are given in Table 1; N-Me peaks are probably methyl resonancesfrom molecules suchasergothioneine,betaine,camitine,and choline;u unassignedresonances CLINICALCHEMISTRY,
Vol.30,No. 3, 1984
=
427
method. By ‘H NMR (integrationof the creatinine CH3 coupling patterns. The general description of NMR spectra is covered in many texts (e.g., reference resonance peak area relative to those of added valine), the creatinine concentration was 9.98 ± 0.36 mmolJL (mean ± 14) and will not be elaborated here. SEM). The corresponding result by the Jaff#{233} method was Creatinine, usually present in urine at millimolar concen10.2 ± 0.1 mmol/L. These NMR values are uncorrected for trations (15), gives rise to two singlet ‘H NMR peaks having any overlap of signals; however, the ratio of the intensities chemical shifts of 3.07 and 4.12 parts per million (ppm) in of creatinine CH3 to CH2 resonances suggested that any the ratio of 3:2, corresponding to the NCH3 and CH2 protons error due to this would be b: 3.94 (s)
113 (0654(c Hl-Kl
a
3-D-Hydroxybutyrate
Bu
H3CNCHO.
aH3C-CH-CH---CO2
a:1.24(d) b: 2.4 (ABX)
48 (0-200)
a: 2.34 (a)
33 (0-67)
70
Hp(,,..Hl
OH Acetoacelate
Acac
Alanine
Ala
a H3C-C--CH2--C02 0
000
+
H3N-CH-C02
a:
1.48(d)
C, lCH2l
13
(0-45) 4.0
CH Lactate
Lac
a:1.34(d)
aH3C-CH--CO2
13
Op ICH2)
(0-78) OH b
Histidino
His
5/mn,
CHa__CO
HN”.CH2-
+NH3
Indoxyl sulfate
2
0I,
a: (I) b: 4.04 7.36(a) C:
ass (a)
3-5
a: 7.19 (dd)
IS
-
b: 7.29 (dd) C: 7.35 (8) d: 7.49 (d) 8: 7.69(d)
0
0-0
0
c,lcila)
Q-o---Q-
3.0
0
Formate
Fm
Dihydroxyacetone
Ha
aHCO2 HO-CH-C--CH-OH
a: 8.48(s)
-
a: 4.43 (s)
-
c
0 c-Ketoglutarate
Kg
O2C-CH-CH--C.--CO2
0
a: 2.48(t) b: 3.02 (t)
8M measured from Fig. 1 for normal human urine at pH 5.6: (s d = doublet, t = triplet, dd
=
tem.ABX = AB partof ABX one value>200.
2.0
singlet, 2nd-order spin sys=
doublet of doublets, AB = spin system). #{176} From 13 normal subjects. 2Only
428 CLINICALCHEMISTRY,Vol. 30, No. 3, 1984
4
#{212}
5 PH.
Fig. 2. Chemical shift vs pH for a titration of a mixture of unne components,containing creatinine(0), citrate (‘C>),hippurate () , and glycine (0), frompH#{176} 3.8 to7.9,monitoredby 1H NMR at200 MHz
Gic
1-Gb
0 -Gic
(a)
2HOH
Acac
Cr Cr
Bu Cl
_____ 5.3
I 4
I 3
2
A08C
Ac
i
6/ppm
1
S/ppm
(b)
i 0Iy
4
3
2
Fig. 3.400MHz 1H NMR spectra of (a) urine from a diabeticpatient, 4 h after voluntaryinsulindeprivation,and (b) unne from a normal malesubject, after 35 h of fasting, showing excretion of ketone bodies and, in a, glucose b has greatfy increasedvertical expansion, i.e., creatinine concentrations are similar in bothspectra.Conditionsas Fig. 1except:(a)48 accumulations,40 #{176}pulse, 0.85-s pulse interval;(b) 493 accumulations, 400 pulse, 1.6-spulse interval.Abbreviations as in Table 1 exceptGIc = glucoseandAc = acetone measure glucose, and to distinguish between the anomeric forms, by noting the two distinct doublet resonances at 5.3
and 4.7 ppm, corresponding to the anomeric protons of aand /3-1)-glucose,respectively. Spectra shown in the Figures are referenced to either glycine (methylene singlet at 3.57 ppm) or hippurate (orthoproton doublet at 7.83 ppm). These resonances do not shift within the normal pH range of urine (pH 5-8), whereas resonances for compounds with plC8 values within this range can shift significantly. Figure 2 shows the result of a pH titration of a model solution containing glycine, hippurate, creatinine, and citrate, monitored with ‘H NMR spectroscopy. The resonances of citrate (which has plC8 values of 3.13, 4.76, and 6.40) (16) and creatinine (PKa of 4.80) are clearly shifted. Figure 1 also illustrates that various organic acid metabolites, traditionally determined by gas chromatography/mass spectrometry (17), are identifiable by this NMR technique: e.g., acetoacetate, 3-u-hydroxybutyrate, and formate. Quantitative measurements by NMR are in a very early stage, and possible interferences with the method have to be considered. For accurate measurement of peak intensities
there must be a sufficiently long relaxation delay, so that the magnetization returns fully to equilibrium between pulses. With a 90 #{176} pulse, this delay should be at least three times T,, where T, is the spin-lattice relaxation time of the proton considered. With smaller pulse angles, however, a more rapid repetition rate can be used. Problems arising from overlap of peaks are more difficult to overcome, although the dispersion of chemical shifts increases with field strength (i.e., 400 MHz is preferable to 200 MHz) and many
have more than one resonance. Resonances may also become severely broadened and, in the extreme case, unobservable if, for example, a small molecule binds to a macromolecule or forms a complex with a paramagnetic ion
molecules
(e.g., cupric or ferric ions). Normal urine contains only small
amounts of macromolecules, but the resonances from these are likely to be broadened, owing to slow tumbling in solution. Also, resonances from chemical species that have highly coupled ‘H spin systems may be hard to detect and assign, particularly for speciesthat are present in relatively low concentrations. Some components
of urine-urea, uric acid, and inorganic phosphates-have either no protons or only NH protons. The latter are sometimes broadened by exchange with water protons and therefore are not amenable to routine study by this method. Other examples of exchangeable protons are those of OH, SH, and COOH groups. Continuous irradiation of the water resonance may lead to cross-saturation with exchangeable protons. Resonances overlapping with water will also be irradiated, and hence other coupled nuclei may also be affected. After lyophilization and on redissolving in 2H20, acidic protons can exchange with deuterium. These include some acidic CH protons such as acetoacetate CH2, and even the creatinine CH2 protons exchange under certain conditions (18), as shown later in Figure 6. The nature of these interferences needs to be further investigated.
Exercise and Fasting The ‘H NMR spectrum of urine collected immediately after hard exercise (Figure 3a) differs from the pre-exercise spectrum in several minor ways. The major difference is a CLINICAL
CHEMISTRY,
Vol. 30, No. 3, 1984
429
Fm
(a)
Methanol
1/
8.5
‘ii’ 4
3
2
Ethanol(CH2)
Ethanol
(b)
Cr
fffl
II
1
/
Cr
/
(CM3)
Hp
fi
Gly
Ci
In’
I
-
4
3
2
1
4.400MHz 1H NMR spectraof (a) urineof a mousethathadingesteddilutemethanolsolution,and (b) normal humanurine,from a subject who had ingested ethanol,showingexcretionof alcoholsand, in a, metabolismof methanolto formate Fig.
Conditions asinFig. 1 except: (a) 532 accumulations, 30 0pulse 4-s pulse interval, 8192datapointszero-filled to 16 384; (b) 48 accumulations,80 0pulse, 1.7-s pulse interval, with suppression of the water resonancewiththe DANTE pulse sequence. Abbreviationsas in Table 1
large increase in the characteristic methyl doublet resonance for lactate (chemical shift of 1.34 ppm) and the associated CH quartet resonance (4.14 ppm). In the ‘H NMR spectrum of pre-exercise urine of this particular individual (not shown), lactate was barely detectable above the background noise, suggesting a concentration of less than 0.1 mmol/L. Fasting in normal subjects results in ketonuria and ketonemia, and it is accompanied by a decrease in blood glucose. Figure 3b shows a spectrum from a urine sample taken after 35 h of fasting, in which ketone bodies and other metabolites could be detected. These observations prompted us to examine urine from diabetic patients.
Diabetes This disease state is associated with abnormally high concentrations of several lOWMr metabolites in plasma and urine, particularly the ketone bodies 3-o-hydroxybutyrate, acetoacetate, and acetone, as well as glucose (19). All of these substances are readily detectable in urine by ‘H NMR spectroscopy, and Figure 3a shows resonances for urine sampled from a diabetic patient 4 h after voluntary insulin deprivation. Approximate concentrations per liter were 3.4 mmol of 3-n-hydroxybutyrate, 3.6 mmol of acetoacetate, and 0.2 mmol of acetone (in control samples these were all 5:1, compared with about 1:1 in urine (Figure 4a). This suggests that relatively more methanol than formate was being reabsorbed from the renal tubular ifitrate. Such results illustrate how the excretion of certain compounds and their metabolites can be simultaneously studied by ‘H NMR scans of urine. As another example, we could readily detect the excretion of ethanol from a normal human subject who had ingested a small quantity of beer (Figure 4b).
Acetaminophen
The convenient use of 1H NMR spectroscopy for the rapid monitoring of the major excretion products after the metabolism of a widely used drug is illustrated in Figure 5. Sixty minutes after the ingestion of 1 g of acetaminophen, three new acetyl resonances appeared in the urinary spectrum and six new doublets (probably three pairs) were observed in the aromatic region. The ratios of these three metabolites varied markedly in urine excreted during the next 2 h. By reference to previous chromatographic studies (20) we tentatively assigned these resonances
to the glucuronide and
‘RlI
II #{149}#{149}fl III
/
Ill
Cr
(a)
Cr 2.2
\
Gly
T
Up
H I 8
I
I 7
,,,,,,.,.
I
4
2
3
i/ppm
(b)
C,
Gly
H
Up
rrip
a
I
I
4
3
6/ppm
2
Fig. 5. A comparisonof the 400 MHz 1HNMR spectraof theurineofa normalsubject (a)60 mEn after and (b) 5 mm beforetheingestion of1 g ofacetaminophen Bothspectraobtained withconditions as in Fig. 1 except:120 aCcumulations and a 4.9-s pulseinterval.Abbreviationsas in Table 1. Three new acetylmethylpeaksand threenew setsof doubletsin the aromaticregionare tentatively assigned to sulfate (I) and glucuronide(II) derivatives, and free drug (Ill). By 98 mm after ingestion resonances forIll weretooweaktobeobserved
a -Gic
0
Gb
Cr
Val
5.3
4
3
2
I
FIg.6. 500 MHz ‘H NMR spectrum of urinefroma man with kidneydamage, showingabnormallyhighconcentrations ofglucoseand amino acids Conditionsas in Fig. 1 except: 512 accumulations, 450 pulse, 5-s pulse interval,32 768 data points; samplewas lyophilized and redissolved in 2H20 so no water suppression was required. Abbreviations as in Table 1 except:GIccontainslargelyresonancesfor glucose, andamino acids are identified by the usualthree-letter codes. Notethat the creatinine CH2 resonancedoes not appear, because of exchange with deuterium (see text)
CLINICAL CHEMISTRY, Vol. 30, No. 3,1984 431
sulfate derivatives of acetaminophen and to the free drug. Further studies involving ‘H NMR decoupling experiments and the analysis of individual metabolites of known structure are in progress. Our current work suggests that these NMR methods will be useful in studies of the metabolism and excretion of other widely used analgesics.
Kidney Damage is slowly excreted by humans and accumulates particularly in the proximal renal tubules. Prolonged exposure to cadmium can result in functional and structural damage to the proximal tubules, accompanied by glycosuria, Cadmium
aniinoaciduria,calciuria, phosphaturia,and, in severecases, excretionof lOWMr proteins and enzymes (21). The ‘H NMR spectrumof urine from a man with known kidney damage, who had worked in the cadmium industry for many years (Figure 6), clearly shows an abnormal metabolite proffle related to defects in renal function-i.e., high concentrations of glucose and amino acids, including alanine, valine, threonine, and lysine. There is currently much interest in the early biochemical detection of renal lesions caused by cadmium exposure. Present indices of exposure, which are usually related to the degree of low-Mr tubular proteinuria,
include particularly
the excretion of f3-microglobulin and retinol-binding protein (21). ‘H NMR spectroscopy can also detect various other abnormal metabolites associated with cadmium intoxication and may provide further about the onset of renal lesions.
information
The examples cited in this paper demonstrate the scope of high-resolution, high-field ‘H NMR spectroscopy in the clinical analysis of urine. For such a complicated mixture of compounds, a large number of species can be identified and, in favorable cases, quantified without separationor derivaFrom our studies so far, we concludethat ‘H NMR
tization.
spectroscopy is a useful technique for the routine study of the excretion of a wide range of metabolites and drugs.
We are very grateful to Rio Tinto Zinc Services,the Arthritis and Rheumatism Council, Science and Engineering Research Council, Medical Research Council,and the University of London Intercollegiate Research Servicesfor support;to Drs. G. Kazantzis (London SchoolofHygiene and Tropical Medicine, London, U.K.), S. Juul, A. Macleod, and P. Sonksen (St. Thomas’s Hospital Medical School, London, U.K.) for supplying patients’ urine samples and for their encouragement throughout this work; and to Dr. J. Timbrell (Schoolof Pharmacy, London, U.K.) for the chemical measurements of urinary creatinine.
References L Daniels A, Williams RJP, Wright PE. Nuclear magnetic reso-
432
CLINICAL CHEMISTRY, Vol. 30, No. 3, 1984
nance studiesofthe adrenal gland and some other organs. Nature (London) 261, 321-323 (1976). 2. Brown FF, Campbell ID, Kuchel PW, Rabenstein DL. Human erythrocyte metabolism studiesby ‘H spin-echoNMR. FEBS Left 82,12-16 (1977). 3. Bock JL.Analysisofserum by high-field proton nuclear magneticresonance.Clin C/tern 28,1873-1877 (1982). 4. Traube M, Bock JL, BoyerJL. o-Lactic acidosis after jejunoileal bypass: Identification of organic anions by nuclear magnetic resonance spectroscopy. Ann Intern Med 98, 171-173 (1983). 5. NicholsonJK, Buckingham MJ, Sadler PJ. High resolution‘H NMR studiesof vertebrate bloodand plasma. Biochem J 211, 605615 (1983). 6. NicholsonJK, O’Flynn M, Sadler PJ, et al. Proton nuclear magnetic resonance studies of serum, plasma and urine from fasting normal and diabetic subjects. Biochem J (1984). In press. 7. Matsushita K, Yoshikawa K, Ohsaka A. ‘H NMR of human urine. JEOL News 18A, 54-56 (1982). 8. iles RA, Buckingham MJ, Hawkes GE. Spin-echo proton nuclear magneticresonancedetection of normal and abnormalmetabolites in plasmaand urine. Biochem Soc Trans 11,374-375(1983). 9. Kimber BJ, Roth K, FeeneyJ. Signal-to-noiseratio in accumulation ofFourier transform nuclearmagneticresonancespectra. Anal Chem 53, 1026-1030 (1981).
10. Morris GA, Freeman R. Selectiveexcitation in Fourier transform nuclear magnetic resonance. J Magn Reson 29, 433-462 (1978). 11. Hahn EL. Spin echoes.Phys Rev 80, 580 (1950). 12. Rabenstein DL, Nakashima TF. Spin-echo Fourier transform nuclear magnetic resonance spectroscopy.Anal Chem 51, 1465A1474A (1979). 13. Tietz NW, Ed. Fundamentals of Clinical Chemistry, 2nd ed., WB Saunders, Philadelphia, PA, 1976,p 996. 14. Jardetzky 0, Roberts GCK. NMR in Molecular Biology, Aca-
demicPress, New York, NY, 1981. 15. Blank DW, Nanji AA. Ketone interference in estimation of urinary creatinine; effect of creatinine clearanceindiabeticketoacidosis.Cliii Biochem 15, 279-280 (1982). 16. Dawson RMC, Elliott DC, Elliott WH, Jones KM, Eds. Data for Biochemical Research, 2nd ed., Oxford UniversityPress,Oxford, U.K., 1972. 17. Witten TA, Levine SP, King JO, Markey SP. Gas chromatographic/mass spectrometric determination of urinary acidprofiles of normal young adults on a controlled diet. Clin C/tern 19, 586-589
(1973). 18. Srinavasan R, Stewart R. The catalysisof protonexchangein creatinine by general acids and general bases.Can J C/rem 53,224231 (1975). 19. Zilva ,JF, Pannall PR Clinical Chemistry in Diagnosis and Treatment, 3rd ed., Lloyd-Luke, London, 1981. 20. Mrochjek JE, Katz S, Christie WH, Dinsmore SR. Acetaminophen metabolism in man, as determined by high-resolution liquid chromatography. Clin C/rem 20, 1086-1096 (1974). 21. FribergL, Piscator M, Norberg GF, KjellstromT. Cadmium in the Environment, CRC Press,Cleveland,OH, 1974.
