15N Sample preparation for protein turnover measurements in large populations

255

Analytrca Chrmtca Acta, 241 (1990) 255-260 Elsevler Science Pubhshers B V . Amsterdam

15N Sample preparation for protein turnover measurements in large populations T Preston

*

Scottuh Umuersrtres Research and Reactor Centre, East Kllbnde, Glasgow G75 OQU (Great Bntarn)

D C McMlllan Untverslty Department of Surgery, Glasgow Royal Infirmary, Glasgow G4 OSF (Great Brrtarn) (Received

19th Apnll990)

Abstract

The major lmutatlons to the adoption of whole body protein turnover measurements to assess protem metabohsm m large groups have been the time-consummg nature of sample preparation and the cost and complexity of mass spectrometnc tracer analysis The mtroductlon of contmuous flow isotope ratlo mass spectrometry has greatly improved sample throughput and reduced costs for 15N analysis m punfled metabolic products, such as ammomum However, punftcatlon of unnary ammomum 1s not sufficiently simple or rapld for the large sample numbers generated Thus study compares separation of unnary ammomum by usmg a new unmoblhsed cation-exchange resin apparatus with Conway nucrodlffuslon, using KHSO, as an acidic ammomum trap that IS compatible with automated combustion analysis The alternative methods gave equally good analytical preclston It IS concluded that rmcrodlffuslon would be supenor m nummally equipped field laboratones, whereas the design and speed of the method using an unmoblhsed catlon resin would make this the method of choice in laboratones with a higher degree of automation Keywords Sample

preparation,

Protein

metabolism,

Unne,

The measurement of whole body protein turnover (WBPT) IS of great value m the mvestlgatlon of protem metabolism m health and disease [l] Stable isotope-labelled (15N or 13C) ammo acids are used to derive WBPT values When combined with conventional mtrogen balance rates of whole body protem measurements, synthesis and breakdown can be calculated Such data has been of great benefit m elucldatmg the mechanism of an alteration m nitrogen balance To date, WBPT measurements have been hrruted to studies of small groups of mdlvlduals, usually fewer than ten There IS a need to study protein metabolism m larger groups of more than one hundred subjects, to establish the protein status and nutritional requirements of a population 0003-2670/90/$03

50

0 1990 - Elsevler

Science Publishers

Protein

turnover

The use of stable isotope tracers IS clearly necessary m WBPT studies of children and m adults of chdd-bearing age A number of protocols have been investigated for such studies, mcludmg those using “N-1abelled glycme [2] of l-‘3C-labelled leucme [3] The former can be accomphshed m a non-invasive way by single or multiple oral tracer doses, with analysis of the tracer ennchment m a urinary endproduct of nitrogen metabohsm [l] The latter IS usually mvaave, and requires more complex analysis to measure the isotope ennchment m plasma leucme and breath carbon dloxlde, and IS therefore less suitable for studies m large SubJect groups The protocol most suited to large population studies was introduced by Waterlow et al [4] This requires mmimal subject cooperation, BV

256

1s used and the a single oral dose of “N-glycme excretion of urmary ammomum 1s analysed for “N ennchment over a 12-h period We mamtam that the cost and complexity of mass spectrometnc analysis and the time-consummg nature of urme sample preparation pnor to isotope analysis have been the major factors hmltmg the wldespread use of tlus protocol The mtroductlon of automated contmuous flow isotope ratio mass spectrometry (CF-IRMS) has greatly reduced the time and complexity of “N analysis m such studies [5] Indeed, the speed of throughput by this technique has transformed the economy of “N and total-mtrogen sample analysis However, sample preparation has remained a bottleneck This work was undertaken to mvestlgate alternative methods of purlfymg urmary ammomum which would comphment the speed and slmphclty of isotope analysis by CF-IRMS The potential of each method to be adapted to analyse urea and ammo mtrogen was also a criterion m then selection The alternatlve preparation methods that were investigated were Conway nncrodlffusion and use of an lmmoblhsed cation-exchange resin These were compared to an established batch cation-exchange method used m our laboratory Ths study reports data on precision of isotope analysis and sample recovery Furthermore, each method 1s demonstrated by calculatmg a WBPT rate

EXPERIMENTAL

Separation of urmary ammomum by mlcrodlffuslon Conway mlcrodlffusslon Ammonium was separated from urme samples by using commercial plastic Conway units (small Bel-Art Conway dlffusion units, Radleys, Essex, Great Britain) Volumes of 50 ~1 2 5 M KHSO, and 250 ~1 18 MOhm water were added to cover the base of the centre well of the dlffuslon umt A 200+1 volume of urine, pH 5 5-7 5, pre-filtered through a 0 2-pm pore 25 mm diameter Luer fitting filter unit (Mllhpore, Watford, Great Bntam) was plpetted mto the middle well of the diffusion umt with 2 ml borate buffer pH 9 8 (1 0 M boric acid saturated

T PRESTON

AND

DC

McMILLAN

with KCl, taken to pH 9 8 with 10 M KOH) A 4-ml volume of the same buffer had been added to the outer well to provide a seal for the hd The hd was fitted with a twist to ensure an effective hquld seal, and the unit was incubated at room temperature without shakmg for a predetermmed penod In our hands, the penod reqmred for full recovery of urmary ammomum was 6 h In this study, samples were mcubated for 7 h, unless otherwise stated Full recovery of sample ammomum could have been obtained more rapidly by use of elevated temperatures and shaking The simpler approach was chosen to provide a method that would allow simultaneous mcubatlons of many tens of samples At the end of the diffusion period, replicate 100~~1 subsamples (approximately 50 pg N) of the KHSO, solution were plpetted mto a prefrozen 250~~1.1tm combustion container and lyophlhsed for storage or immediate isotope analysis [5] Solutions m combustion containers can alternatlvely be oven-dried, however some sample loss can occur if hquld samples flow from the container This has little consequence to accurate isotope analysis, but 1s undesirable m a study reportmg sample recovery The KHSO, trap was used to avoid corrosion of the combustion vessels The acid solution was prepared from new analytical-grade chermcals and stored m a tightly sealed bottle to avoid contammatlon by ammonium Each batch of the acid reagent was checked for ammonium blank Finally, vessels were liberally rinsed m tap water, dilute HCl and then distilled water, before being dried for reuse Culture vessel mlcrodlffuslon Disposable culture vessels (25 ml) (Stenlm, BDH, Poole, Great Bntam) were also used to investigate then potential as rmcrodlffuslon vessels Urine samples of 100 ~1 were plpetted mto the vessels along with 4 ml borate buffer pH 9 8 A 5-mm pre-combusted glass flbre (Whatman GF/C) or paper disc (Whatman No 1) was held by a stamless steel pm, which pierced the vessel’s cap A 5-~1 volume of 2 5 M KHSO, was plpetted onto this disc and the lid was tightly closed The sample and buffer were rmxed, carefully avoiding hquld contacting the acid trap, and the vessel was left on its back to maxlmlse the liquid surface area An alternative

15N SAMPLE

PREPARATION

FOR

PROTEIN

TURNOVER

MEASUREMENTS

3 x 15 mm paper or glass flbre strip, trapped by the vessel’s hd with 5 ~1 acid plpetted onto the end, was also used Prehmmary expenments demonstrated that this system required a considerably longer time for full ammonium recovery (> 24 h) than the Conway umts used above This fmdmg, and the fact that their use would prove more expensive than the Conway umts, outweighed the advantages of the disposable units This was m contrast to our fmdmgs that disposable plastic vessels provide a useful diffusion apparatus for large-volume (60 ml) samples [6] Urmary ammomum separatton by batch catlon exchange Batch resin method The method of Read et al [7] to separate ammonium ions from urme was used ms 1s based on an a batch cation-exchange resin (AG50W-X8, 100-200 mesh, Blo-Rad, Watford, Great Bntam) m sodlum/potasslum form, that speclflcally binds ammomum ions [8] Our sole modlflcatlon was to release bound ammonium from the resin using 2 5 M KHSO,, pnor to plpettmg and freeze-drying samples m contamers for CF-IRMS analysis [5] Immoblhsed resm dusts Blo-Rex 25-mm lmmoblhsed AG50W-X8 resin discs (Blo-Rad, Great Bntam) were used to streamhne sample preparation These discs could be condltloned, converted mto the sodmm/potasslum form and regenerated for reuse m stacks of ten, connected by their Luer fittings Operations detailed below were convemently camed out by attachmg a 60-ml dlsposable plastic synnge (BD Plastlpak, Dublin) to the top of the stack, and connecting the bottom of the stack to a water pump Sample handling was also greatly slmphfled as prefdtratlon (0 2-pm, 25-mm Mllhpore Luer uruts) and loading were carried out simultaneously, using a syrmge If required, multlple sample handling could be accomplished using a sohd phase extraction vacuum mamfold Stacks of 10 Blo-Rex discs were lmtlally condltloned with 40 ml methanol using a gentle vacuum The discs were then washed with 45 ml delomsed water and then 45 ml 0 1 M NaOH, to place them m the sodium form They were washed to neutrality with about 45 ml delomsed water, and then treated with 45 ml 0 2 M sodmm/potasslum

257

phosphate buffer pH 7 4 [7] They were again washed with 45 ml delomsed water, to be used for sample loading or capped and stored moist at 4°C Unne samples of 800 ~1 were loaded onto mdlvldual resin discs via a 0 2-pm prefllter They were then washed through with 1 ml delorused water Sample ammomum was eluted mto a separate tube usmg 800 ~1 2 5 M KHSO, Subsamples of 100 ~1 were plpetted mto prefrozen combustion containers and freeze-dried for storage or immediate analysis Immoblhsed resin discs were washed and regenerated m stacks of 10 using, m turn, 2 X 60 ml methanol, 2 x 60 ml 10 M HCl, 60 ml delomsed water (to neutrality), 45 ml 0 1 M NaOH, 45 ml delorused water (to neutrality), 45 ml 0 2 M Na/K phosphate buffer pH 7 4 They were finally washed with delomsed water for storage or immediate reuse Units have been reused up to ten times without noticeable detenoratlon m performance Standard “urme” Standard “urine” solution for recovery expenments was made by addition of the chlonde salts of the maJor urinary cations (sodium, potassmm, calcmm, magnesium and ammonium) to delomsed water, to a concentration taken from standard tables [9] “N analysis of purified urrnary ammomum by continuous flow isotope ratlo mass spectrometry Samples generated from the methods described above were designed to produce approximately 50 pg N for analysis Indeed, sample requirement for CF-IRMS 1s m an optimal range for the Conway units and lmmoblhsed resin apparatus The batch cation resin method of Read et al [7] typically produced samples with ten times this amount of nitrogen This was readily subsampled for isotope analysis A commercial CF-IRMS apparatus (ROBOPREP sample convertor and TRACERMASS mass spectrometer, Europa Scientific Crewe, Great Bntam) was used for isotope analysis The process of automatic CF-IRMS analysis of metabolic endproducts has been described previously [5] Ana-

T PRESTON

25X TABLE

1

TABLE

15N Analysis of ammomum from standard unne (“N and total nitrogen analysis was performed by CF-IRMS, on samples of approximately 50 ~.rgN Recovery was calculated from total nitrogen data, and expressed as a percentage of the quantity of ammomum m the ongnal standard unne solution)

Atom% “N SD, n=6 Recovery, %

Standard unne

Conway units

Blo-Rex disc

Batch resin

0 36206 0 00078 100

0 36160 0 00029 106+4

0 36292 0 00079 91+13

0 36949 0 00014 = 93k6

a Analysis represents between-sample preclslon, wtthm-sample preclslon (1 S D , n = 6) was 0 000071 atom% 15N

lytlcal precision 1s described below Sample throughput was about 12 analyses per hour Whole body protem turnover measurement usmg “N-glycme To demonstrate the sample preparation methods the “smgle-shot” tracer method of Waterlow et al [4] was followed A volunteer underwent an overnight fast and received a single oral tracer dose (1 mg “N-glycme per kg body weight), lmmediately after voiding a basehne urme sample Urme samples were collected contmuously for 12 h and pooled Only drmkmg water was taken during this time WBPT rate was calculated using the equations of Waterlow et al [4], from measurements of tracer enrichment m urinary ammonium and the known tracer addition

RESULTS

Table 1 shows the recovery of ammomum from standard urine and the precision of isotope analysis The Conway units gave a recovery of 106% and the lmmoblhsed resin 91%, these compared with 93% from the batch resin method Betweensample reproduclblhty was compared to wlthmsample reproduclblhty m the batch resin method, the former bemg *O 00017 atom% “N, whereas the latter was shown to be f0 00007 atom% “N The accuracy of isotope analysis (n = 6) after purlflcatlon by Conway nucrodlffuslon was compared under circumstances where sample recovery

AND

DC

McMlLLAN

2

Urine analysts from WBPT expenment [“N analysis of urmary ammomum punfted by the methods described m the text Baseline urme samples were taken before gnmg a tracer dose of “N glycme (1 mg “N glycme per kg body we&t) The ennchment m the ammomum endproduct was measured on pooled unne samples, passed over a twelve hour period] Conway umts

Blo-Rex discs

Batch resin

Basehne atom% SD, n=6 Endproduct atom% SD, n=6

0 0 0 0

0 0 0 0

0 36741 a 000011 0 40991 a 0 00295

Atom% “N

0 04377

excess

37104 00069 41481 00249

37326 00051 41515 00351

0 04189

0 04250

d These samples were analysed at a different others, usmg a different instrument cahbratton

time from

the

was deliberately poor Incomplete recovery was achieved by sampling the nucrodlffuslon acid trap after 10 mm (59% ammomum recovery) The enrichment of ammomum after mcomplete recovery (0 39742 atom% “N) was considerably less than that after full recovery (after 7 h, 0 41481 atom% I5 N), mdlcatmg that 14NH, diffuses more rapidly than “NH, This experiment confirms the fmdmgs of other workers, that full ammomum recovery is essential for accurate isotope analysis

[10,111 Table 2 shows the results from the baseline and “N-enrIched urine samples, and Table 3 shows the WBPT rates derived from these data The precision of isotope analysis of the unenriched urinary ammomum was better than *O 0007 atom% “N for all three separation methods, and that of ennched ammonium analysis was between 0 0025 and 0 0035 atom% “N WBPT rates calcu-

TABLE

3

Whole body protem turnover rate (WBPT rates were calculated from the formula of Waterlow et al [4])

m Table

2, usmg

“NtS

Blo-Rex umts

Batch resm

2 82 0 16

2 96 0 27

2 91 0 20

Conway

g Protein per kg weight per day SD, n=6

the data

15N SAMPLE

PREPARATION

FOR

PROTEIN

lated using the three sample were very smular

TURNOVER

preparation

259

MEASUREMENTS

methods

DISCUSSION

The am of tlus study was to compare methods that faclhtate ammomum separation from urme for 15N analysis It was argued that this 1s a maJor stage that currently louts sample throughput m WBPT studies m large population groups Steam dlstlllatlon and aeration methods were previously discounted a\ they both require an apparatus that does not lend itself to hundreds of sample analyses in parallel In contrast, Conway nucrodlffuslon uses a simple apparatus Many dlffusion umts can be set up m parallel They provide a robust and econonucal method of ammonium analysis that can be handled by unskilled operators under field condltlons The reagents chosen for diffusion analysis reflected this reqmrement, m that a borate buffer pH 9 8 was used m place of K&O, to mmlrmse alkaline hydrolysis of arnmes [12] The need for full sample recovery to obtain accurate isotope analysis was confirmed Potassium 1s the preferred cation m alkahne reagents used for nucrodlffuslon [13] Furthermore, use of a hquld buffer reagent and a liquid hd seal on the vessel faclhtates cleaning for reuse The novel use of KHSO, as acid trap for nucrodlffuslon [6] perrmts direct total mtrogen and 15N analysis by CF-IRMS, as it does not corrode the combustion containers Overall, Conway rmcrodlffusion analysis should prove an economc, robust and rehable sample preparation technique for “N analysis m WBPT studle\ m large populations Cation exchange has proven a rapid alternative to Conway n-ucrodlffuslon It 1s a particularly useful technique m circumstances, such as ammomum separation from blood plasma [8], where alkaline hydrolysis of ammes present m high concentrations can potentially interfere with ammonium analysis In the apphcatlon described here, it 1s the speed of analysis that 1s particularly attractive Sufficient exchange capacity was chosen (many times the theoretical requirement to separate ammomum from urine) to avoid mcomplete recovery, potentially resulting in isotope

fractlonatlon Indeed, m prehmmary experiments, sample recovery and isotope analysis precision was unaltered after loading the resm with three times the sample quantity described above Generally, ammomum recovery was more vanable using cation exchange compared to nucrodlffuslon There was, however, no mdlcatlon that isotope fractlonatlon occurred The batch resin method of Read et al [7] proved more tedious than that using the new lmmoblhsed resin discs The additional mltlal expense of these discs 1s offset by then ease of regeneration and reuse Indeed, although not demonstrated m this prehmmary study, automated solid phase extraction apparatus should readily be adapted to further slmphfy their use It should also be stressed that none of the sample preparation methods described here need be conducted m the isotope analysis facility Samples prepared m combustion containers for CFIRMS analysis can readily be transported, without special precautions, to central service laboratories for rapid and economical analysis [5,6] In conclusion, precise analysis of urinary ammonium has been accomplished by using Conway rmcrodlffuslon and a new lmmoblhsed catlon-exchange resin disc In circumstances which require large numbers of analyses of 15N m urinary ammonmm, such as WBPT studies on large populations, the former method 1s more suited to sample preparation m unskilled hands and the latter method 1s more suited to the laboratory with a high degree of automation Either method can be used m a laboratory remote from the isotope analysis faclhty The authors wish to thank Professor J Shepherd for access to the mass spectrometry facility m the Department of Clmlcal Blochemlstry, Glasgow Royal Infirmary

REFERENCES

1 J C Waterlow, P J Garhck and D J Mdlward, Protem Turnover m Mammahan Tissues and m the Whole Body, North Holland, Amsterdam, 1978 2 D Pxou and T Taylor-Roberts, Chn SC], 36 (1969) 283 3 D E Matthews, K J Motd, D K Rohrbaugh, J F Burke,

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4 5 6 7 8

V R Young and D M Bier, Am J Physlol, 238 (1980) E413 J C Waterlow, M H N Golden and P J Garhck, Am J Physlol , 235 (1978) El65 T Preston and DC McMlllan, Blamed Environ Mass Spectrom , 16 (1988) 229 P D Brooks, J M Stark, B B McInteer and T Preston, Sod Scl Sot Am J, 53 (1989) 1707 W W C Read, R A Harnson and D Halhday, Anal BIOthem, 123 (1982) 249 J H Hutchmson and D H Labby, J Lab Chn Med, 60 (1962) 170

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DC

McMILLAN

9 C Lentner (Ed ), Geigy Sclentlfx. Tables, Vol 1, CubaGeigy, Basle, 1981, p 58 10 WA O’Deen and L K Porter, Anal Chem ,51 (1979) 586 11 G L Turner and F J Bergensen, m A H Gibson and W E Newton (Eds ), Current Perspectives m Nitrogen Flxatlon, Elsevler, Amsterdam, 1980, p 482 12 D Sehgson and K Hlrahara, J Lab Chn Med , 49 (1957) 962 13 E J Conway, Mlcrodlffuslon and Volumetnc Lockwood and Son, London, 1950

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