MAGNETIC RESONANCE IN MEDICINE 5, 572-577 (1987)

Indirect Monitoring of Carbon-13 Metabolism with NMR: Analysis of Perfusate with a Closed-Loop Flow System

DANIEL J. OLEARY, SUSAN P. HAWKES,* AND CHAS. G. WADE?

IBM Instruments, Inc., 40 Airport Parkway, San Jose, California 951 10, and *Department of Pharmaceulical Chemistry, University of California, Sun Francisco, California 941 02

Received May 19, 1987

NMR studies of living cells and of perfused organs almost invariably require a major effort in the design of either the probe or the support system. For enriched I3C studies of metabolism, it is sometimes possible to use a simpler approach, one amenable to use in narrow-bore high-resolution NMR spectrometers. Since some of the metabolic changes are reflected by changes in the chemical composition of the media, it may be necessary to provide only for NMR measurements of the perfusate. A simple apparatus is described which allows NMR measurement of the perfusate. A commercially available concentric NMR tube, modified to incorporate flow, is inserted in the flow loop and placed in the spectrometer. An example is given of the metabolism of 13C-enriched glucose to lactate by chick embryo fibroblasts grown in a hollow fiber bundle assembly. o 1987 Academic Press, Inc.

NMR studies of living cells and of perfused organs almost invariably require a major effort in the design of either the probe or the support system ( I ) . Eukaryotic systems that have been studied include cell suspensions (2), anchorage-dependent cell cultures (3-7), and perfused organs ( I c , Id, 8). Each application has a unique system to maintain cell viability during the course of the NMR studies. This emphasis on hardware design is focused upon maintaining the organisms in the spectrometer. For 31P NMR, this emphasis is necessary since the reactions of interest, such as ATP dynamics, occur only in the organism or organ being studied. We wish to point out that for many enriched I3C metabolic studies it is sometimes possible to avoid the use of such refined experimental methods. Since many of the metabolic processes are reflected by changes in the chemical composition of the perfusate, it may be necessary to provide only for NMR measurements of the perfusate. When this approach is applicable, it can greatly simplify the experiment. Although this idea appears to be an obvious one, it has not, to our knowledge, been utilized in NMR studies of living systems. We describe an apparatus made from inexpensive, commercially available components which can be used with most high-resolution NMR spectrometers to follow I3C metabolism in cells and organs. We demonstrate the utility of this approach in 13C NMR of intact, anchorage-dependent cells.

The use of anchorage-dependent cells for NMR studies presents a challenge because the growth surface decreases the filling factor and hence the sensitivity. At least four

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INDIRECT CARBON-I 3 NMR OF PERFUSATE 573

assemblies have been reported (3 , 5- 7) for NMR studies of these cells. One particularly successful approach is the growth of cultured cells on the outer surfaces of porous, hollow fibers (9). Media flows through the center of the fibers and diffuses through the walls to maintain cell viability. The only assembly of this type which is commercially available is the Amicon Vitafiber unit (10). Its use for NMR studies (3) is limited to electromagnets because of its geometry and size and furthermore, it requires modifi- cation of the probe. To circumvent this problem we have designed an assembly which incorporates a bundle of Amicon 3s-100 fibers (outer diameter approximately 200 pm) into a unit which can be inserted into a 15-mm probe of a superconducting magnet (7).

In metabolic studies using isotopically enriched substrates, the media can be a major contributor to spectral information. This article describes a media loop system in Fig. 1 which provides direct observation of NMR signals from the media utilized by cultured cells. The cells can be grown in an Amicon Vitafiber unit or in an assembly of choice. Shown in Fig. 1 is the option of using a probe-compatible assembly (7) which allows for observation of NMR signals from the cells in addition to signals from the media. A recirculating system, a reservoir (1 25 ml Erlenmeyer flask), and a peristaltic pump provide media flow. The system is maintained in a humidified incubator which pro-

Hollow fiber assembly

FIG. 1 . Closed-loop flow system for NMR studies of intact cells. Bypass tubes are used when components are to be excluded from flow.

574 OLEARY, HAWKES, AND WADE

vides a 95% air, 5% C02 atmosphere. Inside the incubator, the media, which is buffered with bicarbonate, travels through approximately 9 ft of gas-permeable Silastic (1 1) tubing (4-in. 0.d. X &-in. id.) which serves to maintain the pH of the media in the assembly. As indicated in Fig. 1, this coil can be bypassed during the time the system is removed from the incubator, such as for media changes and other manipulations which are performed in the sterile environment of a laminar-flow hood. Outside the incubator, media flows through Hytrel BXX lined PVC tubing (12) which is imperme- able to gases and thus maintains the pH of the media during flow outside the COz- enriched atmosphere.

Unique to the studies reported here is the media loop tube, shown in more detail in Fig. 2. A commercial, 10-mm coaxial NMR tube (13) is modified with the addition of three orifices, one at the very tip and two placed on the tapered portion (Fig. 2).

Inlet

Inlet -

Hyirel Bxx lined PVC tubing, 118" 0.d. x 1 / 1 6 i.d.

Silastic medical adhesive plug

10mm NMR tube

Silastic medical adhesive

lOmm co-axial reference

FIG. 2. NMR media loop tube.

INDIRECT CARBON- 13 NMR OF PERFUSATE 575

These are generated with a flame-heated tungsten filament. A 25-cm length of Hytrel tubing inlet line is inserted down the coaxial tube and sealed in the lower neck with Silastic silicone medical adhesive (11). This adhesive is then applied in a thin film on the exterior surface of the coaxial tube, 1 cm above the tapered portion. The coaxial tube is then inserted and sealed in a 10-mm NMR tube. A 25-cm length of Hytrel (outlet line) is inserted into the coaxial tube as shown, and both Hytrel tubes are then sealed into place with Silastic medical adhesive. Media flow is through the inlet tube to the bottom of the NMR tube, upward through the orifices in the taper, then back to the reservoir via the outlet tubing. The tube is supported in the spectrometer with a conventional NMR spinner. It does not spin but the field can be easily shimmed on the free induction decay to proton linewidths of 7 Hz.

All components in Figs. 1 and 2 which are in contact with the media can be au- toclaved for sterile operation. The 25-ml flask shown in Fig. 1 is used for the intro- duction of isotopically enriched substrates for metabolism studies. The larger flask can be bypassed and the smaller 25-ml flask used to limit the amount of enriched substrate required. An injection port allows quick purging of the system prior to isotope studies, so that dilution effects can be eliminated. Purging is accomplished with a sterile 50-ml disposable syringe. The injection port also allows modification of the media in situ. The injection port is a 1 X 1 X 2-cm Lexan piece with tubing connectors at both ends. Each port incorporates a 5-mm HPLC injection septum. Temperature control beyond the incubator is maintained by use of an insulated, temperature-con- trolled conduit which runs from the incubator to the magnet and a variable temperature unit on the NMR.

Carbon- 13 NMR in intact, anchorage-dependent cells serves to illustrate the use of the media tube. Tertiary chicken embryo fibroblasts were seeded and grown in an Amicon assembly at 41°C. The cells were maintained with medium 199 (GIBCO), supplemented with 2% tryptose phosphate broth, 2% calf serum, and 1% chicken serum. The glucose content of this preparation is 0.1% (w/v). At zero time, the system was purged of this media and replaced with media (14) identical in all respects except the unlabeled glucose was replaced with an equal amount of uniformly labeled ['3C]glucose of 97% isotopic purity (15). NMR spectra of the media were then acquired every 30 min for a 10-h period. Data were acquired at 50 MHz on an IBM Instruments AF200 spectrometer equipped with a 10-mm VSP tunable broadband probe and a BVT- 1000 variable temperature unit. Spectra were obtained in the unlocked mode with WALTZ proton decoupling, and the @-glucose C-1 peak at 96 ppm served as an internal reference. Figure 3 shows a stacked plot of the region 0-66 ppm. The peak at 60 ppm is from the C-6 carbons of both a and @ anomers of glucose. Under the experimental conditions used, the end product of metabolism is lactate, and the signal from the lactate methyl doublet is at 2 1 ppm. Each spectrum required 20 min acqui- sition time.

The data allow construction of substrate utilization/product formation curves suit- able for comparison under a variety of conditions. During the experiments, the cell culture is undisturbed.

Indirect monitoring of I3C NMR using the media tube provides a simple, inexpensive method of following metabolism. When combined with commercial cell-growing as- semblies, such as the Amicon Vitafiber unit, the indirect monitoring method allows

576 O’LEARY, HAWKES, AND WADE

-10

66 0

PPM

FIG. 3. Stacked plot of I3C spectra of culture media initially 0.1% (w/v) [U-13C]glucose. Each spectrum consists of 1860 transients, acquired using 60” pulses (7.7 ps) with an 11.6-kHz sweep width and 16K data points. Pulse spacing was 0.7 s.

studies of carbon metabolism in intact cells without extensive design effort for probes or support systems. Since the media loop tube can be shimmed to good resolution and used with proton decoupling, sensitivity is high. It should be emphasized, however, that this method is applicable only to metabolites not localized inside the cell or organ. Indirect monitoring could easily be extended to other cell systems and to perfused organs. In fact, some variation of this method may be an advantage in enriched ”C studies of perfused organs. Since the I3C signals from the media are invariably measured concurrently with those of the organ, analysis of the perfusate with a closed-loop flow system, as described herein, would provide a method of distinguishing the two signals.

ACKNOWLEDGMENTS

The authors thank Mark ONeil-Johnson and Leo Pallanck for their assistance. The financial support of IBM Instruments, Inc., and the Elsa Pardee Foundation (to S.P.H.) are gratefully acknowledged.

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INDIRECT CARBON-1 3 NMR OF PERFUSATE 577

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