Measurements of triiodothyronine and thyroxine in human serum by gas-liquid chromatography

ANALYTICAL

43, 43&-445

BIOCHEMISTRY

Measurements Human

of Serum

NORIYUKI TERUNORI ANN Endocrine New

(1971)

Triiodothyronine by Gas-liquid

and

Thyroxine

in

Chromatography’

N. NIHEI,* MARVIN C. GERSHENGORN,s MITSUMA; L. RENEE STRINGHAM, CORDY, BARBARA KUCHMY, AND CHARLES S. HOLLANDER”

Unit, Department of Medicine, New ITork University School of Metlicittr. York, New York 10016, and Endocrine Cnits, Department of Medicinr, University of Rochester School of Medic&e at Strong Menwrial ad Rochester General Hospitals, Rochester, Xew IYork l.$f:i’l Recrived

January

27. 1971

Gas-liquid chromatography (GLC) has been suggested as a method for measuring iodinated amino acids (l-3). Theoretical advantages include great sensitivity, specificity, and versatility. Picogram sensitivity to halogens is attainable with the electron capture detector (4). Specificity results from the high resolving power of GLC (5) and versatility stems from the fact that chromatographic reparation permits simultaneous analysis of a number of iodinated amino acids. The technique of GLC has been applied to the analysis of chemically pure standards of iodoamino acids. Stouffer et al. (1,2), and shortly thereafter Richards and Mason (3) succeeded in applying GLC to the measurement of nanogram quantities of 3,5,3’-triiodo+thyronine (T,) and L-thyroxine (T4), the normal secretory products of the thyroid gland. More recently a number of workers, including Alexander and Scheig (61, Shahrokhi and Gehrke (7)) and Funakoshi and Cahnman (8’) : ‘Presented in part before the annual meeting of the Association of American Physicians, 1968, and the annual meeting of the American Thyroid Association, 1968. ’ Formerly Fellow in Endocrinology Rochester General Hospital and University of Rochester and later Instructor in Medicine, N. Y. U. School of Medicine. Currently Chief of Endocrine Division First Department of Internal Medicine, Nagoya University School of Medicine, Japan. 3 Predoctoral Fellow, N. Y. U. School of Medicine. ‘Fellow in Endocrinology and Instructor in Medicine, N. Y. U. School of Medicine. ‘Formerly Chief of Endocrine Unit Rochester General Hospital, Assistant Professor of Medicine, University of Rochester School of Medicine. Currently Chief of Endocrine Unit and Director of Clinical Research Unit, Associate Professor of Medicine, N. Y. U. School of Medicine. 433

434

NIHEI

ET AL.

employed trimethylsilyl derivatives for the characterization of iodoamino acids by GLC. None of these workers has been able to perform serum determinations by GLC because of difficulties with extraction of the iodothyronines and purification of the derivatives. Recently Volpert (9) described an elaborate method for the purification of these derivatives. We describe here a sensitive gas chromatographic method for the measurement of both T, and T, in human serum. The procedure involves: (1) extraction of the iodothyronines from serum in a form suitable for derivative formation; (2) a simple method for the purification of this derivative with a weakly alkaline anion-exchange resin; and (3) the use of temperature programming to eliminate extraneous substances and permit quantitation of T, and T,. Although T, was discovered in 1952 (10) and has been shown to have a higher metabolic potency, a more rapid turnover, and a larger volume of distribution than T,, implying a major role for it in thyroid hormone calorigenicity (11)) measurements of its actual concentration in human serum have not been documented. Estimates of serum T, levels in normal subjects by a number of workers using a variety of techniques (12-21, 23-31) have varied over a nine-fold range. This paper describes GLC measurements of serum T, in a number of thyroidal states. MATERIALS

Pivalic anhydride (Distillation Products Industries, Eastman Kodak Co., Rochester, N. Y.) : Upon receipt, it is divided into 5 ml portions, placed in small brown bottles, and stored in a desiccator at -30°C. The reagent has a safe shelf-life of 30 days. Triethylamine (Distillation Products) : Technical grade; redistilled prior to use. Methanol (Mallinckrodt Chemical Works, St. Louis, MO.) : Spectral grade. Benzene (Mallinckrodt) : Spectral grade. Methyl&on mixture: Absolute methanol is saturated with dry hydrochloric acid gas (Mallinckrodt). It is prepared once every 6 months and is stored in a brown bottle at -30°C. Pivalylation mixture: Consists of pivalic anhydride, triethylamine, and methanol in a ratio of 20: 1: 1. It is prepared freshly within 1 hr prior to use. Nitrogen drying apparatus, N EVAP (Organomation Associates, Shrewsbury, Mass.) : It was modified by removing the steel needles provided by the manufacturer and substituting disposable Pasteur pipets which are attached with a 4 cm length of rubber tubing.

SERUM

T:$ AND

T., BY GLC

435

Plastic Identi-plugs (Gaymar Industries, Buffalo, N. Y.) . L-Thyroxine (T4), 3,5,3’-triiodo-L-thyronine (T3), and 3,5&iodothyronine (T2) (Sigma Chemical Co., St. Louis, MO.) : Obtained as free acid in the DL form and chromatographically purified prior to use (see below). 3,3’,5’-triiodo-L-thyroxine (RT,) : This compound was a gift from Dr. Meltzer at Warner-Lambert Laboratories. Thyroxine 1311 and triiodothyronine 1~~1 (Abbott Laboratories, North Chicago, Ill.) : Stored at -30” immediately upon receipt and chromatographically purified prior to use (see below). Bio-Rad resin AG 5OW-X2 1004200 mesh in the hydrogen form (BioRad Laboratories, Richmond, Calif.) : Analytical-grade cation resin. Amberlite IR 45 ion-exchange resin (Mallinckrodt) : Analytical reagent. Disposable glass columns (Rochester Scientific Co., Rochester, N. Y.) : Fabricated to the specifications described. 5% OV-I on Chromosorb WHP (Supelco, Inc., Bellefonte, Pa.). METHODS The GLC procedure for the measurement of iodothyronines in serum consists of five fundamental steps: (1) extraction of T, and T, from serum by passage through a cation-exchange resin column; (9) preparation of stable volatile derivatives; (3) purification of the derivatives with an anion-exchange resin column; (4) gas chromatographic separation and quantitation; and (5) correction for methodological conversion (deiodination) of T, to T,. (I) Extraction. Tracer quantities of T, lZRI and T, 1311were added to a 1.1 ml sample of serum and a 0.1 ml aliquot retained to permit calculation of recovery. A maximum of 16,000 disintegrations per minute of each isotope was added. The isotopes were stored in a freezer at -30°C and were purified prior to use by five successive separations in a onedimensional paper chromatography system using a t-amyl alcohol/ hexane/ammonia solvent (21). This number of separations was required to achieve uniform specific activity in other purification experiments (see below). A cation-exchange resin (AR) AG 5OW-X2 100-200 mesh in the H+ form (21-23) was washed and soaked in distilled water overnight. A disposable glass column 25 X 0.6 cm i.d. with a drawn out 7 cm portion at the base was fitted with a glass wool plug at the point of the column narrowing. The resin was poured on to the column to a height of 6 cm and then purified by washing successively with: 10 ml of distilled water, 3 ml of 1 N HCl, and 6 ml of distilled water. Then 1 ml of labeled serum was added to the column and washed with 15 ml of 0.15 M ammonium acetate, pH 8.5, to remove most of the lipids and proteins.

436

NIHEI

ET

AL.

The iodothyronines were then eluted with 6 ml of 3.4N N&OH. The first milliliter was discarded and the next 5 ml of eluate, which contained most of the T, and T, in the serum, was collected and dried under nitrogen. (2) Preparation of the Derivative. Prior to derivation of the standards, free acids of T, and T, were chromatographically purified in a onedimensional paper chromatography system using a t-amyl alcohol/ hexane/ammonia (5: 1:6) solvent. Tracer quantities of 1311-labeled acids were added so that specific activity could be determined after each separation. The specific activity was shown to be constant, assuring purity of the material, after the fourth and fifth chromatographic separations. In the case of T, a tracer quantity of T, lZ51 was added at the outset and the final T, preparation was virtually devoid of lZ51 countsi.e., contained less than 0.02% of the lZ51 counts added. Elution from paper was accomplished with a methanol/acetic solution (5 parts absolute methanol to 1 part 1 N acetic acid). The methyl-N,O-dipivalyl derivatives were prepared by the method of Stouffer (1,2) with minor modifications. The methylation was performed by adding 0.25 ml of a solution of absolute methanol saturated with dry hydrochloric acid gas to the dried extract or dried sample. The samples were then sealed under dry nitrogen, capped with Teflon tape and a screw cap, mixed by vortex, and heated to 70°C for 10 min in a water shaker bath. The tubes were then immersed in a 40” water bath and dried under a dry nitrogen steam. The sample was acylated by the addition of 1 ml of a freshly prepared solution containing pivalic anhydride, absolute methanol and freshly distilled triethylamine in a ratio of 20: 1: 1. The reaction mixture was then sealed, mixed, heated, and dried as described for methylation except that it was incubated at 70” for 30 min. (3) Purification of the Derivative. Amberlite IR 45 anion-exchange resin in the hydroxide form was washed successively with four times its volume of absolute methanol and then benzene. The resin was poured into a 25 x 0.6 cm i.d. glass column to a height of 7 cm and settled with 25 ml of benzene. The sample was dissolved in 1 ml of benzene and applied to the resin column. The bottom of the derivative tube was washed with three 0.5 ml vol of benzene, which were passed through the column. A final 1.5 ml of benzene was added directly to the column. The effluent was collected in a 13 X 120 mm counting tube and dried under nitrogen and yield determined in a gamma counting spectrometer (NuclearChicago Corp.). (4) Gus Chromutogrup.hic Separation and Quuntitation. The MicroTek MT 220 gas chromatograph (Tracer, Inc., Austin, Tex.) used in

SERUM

T3 AKD

T4 BT

GLC

437

these studies was equipped with a 63-nickel electron capture detector having a background current of 5 X lCPg amp. Detectors with a lower background current were not suitable for serum analysi,s. 2 ft U-shaped glass columns (4 mm i.d., 6 mm o.d.) were packed with 5% OV-1 on Chromoeorb WHP (Supelco) to 1.5 cm from either end and conditioned for 16 hr at 300°C with a carrier gas (10% methane, 90% argon) flow of 110 cc/min. Samples were dissolved in 100 ~1 of benzene and 5 ~1 was injected directly on the column. After a 12 min initial hold at 220”, temperature was programmed at 3”Jmin from 220°C to 300°C with a final temperature hold of 5 min. Other operating parameters included: inlet temperature of 280”; detector temperature of 350°C; external electron capture detector power supply (model 11538-000) settings-RF mode, pulse rate 1.1 X 104/sec, and pulse width 0.5 psec with an electrometer attenuation of lo2 X 64. The stability of the system was assessed by repeated analysis of 5 ~1 aliquots of a standard solution that contained 1 ng of T, and 2 ng of T,. This calibration standard was injected under the identical conditions and immediately following each sample. For quantitation of a sample peak the slight variation in column and/or detector performance from one analysis to the next was compensated for by comparing its area to that of the calibration standard. The peak areas, i.e., the integrated area under each peak on the chromatographic tracing, were measured with an electronic integrator (Infotronics digital readout model No. CRS-100, Infotronics, Inc., Houston, Texas) and the T, and T, concentrations determined using standard curves. The system response was initially standardized by analyzing solutions containing increasing amounts of T, and T, added to serum from an untreated athyreotic subject. Both hormones were prepurified by five successive paper chromatographic separations in t-amyl alcohol/hexane/ ammonia prior to use. Two separate curves were plotted for each hormone. For the first set of curves, corresponding to the hypothyroid range, the final hormone concentrations in nanograms per 5 ,~l varied from 0.025 to 0.125 for T, and from 0.1 to 0.5 for T,. For the second set, corresponding to normal and hyperthyroid concentrations, the final hormonal levels ranged from 1.25 to 2.5 ng per 5 ,~l for T, and from 0.5 to 15 ng per 5 ~1 for T,. The quantity injected, corrected for recovery through the entire procedure and for the T, and T, present in the blank-hypothyroid serum alone, was plotted against its peak area on the chromatographic tracing. In practice, the standard curves represent a plot of the T, or T, concentration of the original serum sample versus the ratio of the peak area of the sample and its calibration standard. (5) Conversion. It was observed that, when a known amount of T,

438

NIHEI

ET

AL.

was added to a serum sample, a small proportion was converted to T, during the process of extraction and measurement. The extent of this methodological conversion was assessed by adding known amounts of T, to hypothyroid serum, measuring the resultant T, concentration, and correcting for the small amount of T, originally present in the T, standard and in the blank (hypothyroid serum alone). Clinical

Material

All subjects were given careful clinical and laboratory evaluations and only the cases that fulfilled the criteria described here were selected for study. The normal group included subjects who were found to be euthyroid after a history and physical examination, had no palpable enlargement of the thyroid gland, and had a normal PBI or T, by MurphyPattGe and a normal free T, determination. Hyperthyroid patients were selected after a history and physical examination, and had an elevated PBI or T, by Murphy-Pattee and high free T, and an abnormally increased thyroidal uptake of radioiodine. All had toxic diffuse goiter, i.e., a diffusely enlarged thyroid gland with no nodules on palpation and a diffuse uptake of radioiodine on scan. Patients were considered to be hypothyroid after a history and physical examination and abnormally low PBI or T, by Murphy-Pattie, free T4 and radioactive iodine uptake determinations. RESULTS

Methodological

Results

1. Standard Curves. The standard curves for both T, and T, were constructed with multiple measurements. Analysis of these curves revealed that they could have been represented, over a very wide range, as single hyperbolic functions. Within narrow limits it was found that they were linear, using the “least-squares” method of analysis with coefficients of correlation above 0.99. The response to both T, and T, was linear for concentrations of T, to 274 ng per 100 ml with a curve formulated as y = 20.1~ - 0.2 and for T, to 1 pg per 100 ml with a curve of y = 39.8x + 0.3. Above these values linearity is retained to maxima of 5 pg per 100 ml for T, and 30 ,ug per 100 ml for T,, but with curves formulated as y = 13.8~ + 0.7 for T, and y = 36.7x + 3.9 for Tq, respectively (Fig. la,b) . 2. Chromatographic Tracing. An analytical tracing of normal human serum is shown in Fig. 2. The steady baseline, with recovery to virtually preinjection levels and complete resolution of the T, and T, peaks, facilitates quantitation.

SERUM

T4

rig/ml ,-/

Ts AND

439

Tq BY C&C

25

501

T3 rig/m! .--9

I’ -LI

l--L 0.1

-105 1

1-0.2

03

INTEGRATOR UNITS 'INTEGRATOR

UNITS OF STANDARD

(B)

10 20 30 INTEGRATOR UNITS

40

5.0

'INTEGRATORUNITS

6.0

7.0

8.0

OF STANDARD

Fro 1. Standard curves for serum measurement of T, and T.,. Values have been plotted as rig/ml versus ratio of integrator units of the sample to integrator units in a standard solution containing 1 ng T3 and 2 ng T,. All curves have been drawn by the method of “least squares” with coefficients of correlation great.er than 0.99 in each case. (a) For serum T3 measurements between 0 and 274 ng per 100 ml and T, between 0 and 1.0 pg per 100 ml. The equations for these curves are v= 20.12 - 0.2 for T, and g = 39.82 + 0.3 for T,. For adequate visualization the axes have been considerably expanded, exaggerating the deviation of the plotted points. (b) For serum T, measurements above 274 ng per 100 ml and T, above 1.0 pg per 100 ml. The equat,ions for these curves are ?J= 13.&+0.7 for T, and y=36.72+3.9 for T,.

3. Determination of the Conversion Ratio. Exogenous T, added to a serum sample prior to analysis falsely elevates the T, measured. Despite careful precautions, approximately l-276 of the T, present in a serum sample is converted (deiodinated) to T, during the analytical procedure. This methodological conversion, although insignificant when measuring T,, must be accurately assessedin order to meaningfully determine the

440

NIHEI

ET AL.

ii $ % E oz x 8 Y

A SOLVENT

FRONT

il

1L _J T4

\ A TEMPERATURE PROGRAM

MINUTES

AFTER

INJECTION

FIG. 2. Gas chromatogram of serum sample: tracing of derivatized serum extract from a normal subject. The isothermal portion of the program, which ends at the arrowhead, is 12 min long and the Ta and T, retention times were 33 and 38 min, respectively. A calibration standard has been injected immediately following the sample (see text). In this case, a 23 min portion of the tracing has been deleted.

comparatively small endogenous T, in serum. Therefore, purified T, was added to hypothyroid serum known by prior measurement to contain less than 0.3 pg of T, per 100 ml and less than 0.05 ,ag of T, per 100 ml. The extent of conversion was determined by measuring T, and T, in the enriched serum and correcting for the small amount of T, and T, found to be present in the blank. A priori, the possibility that the conversion ratio varies with the T, concentration could not be excluded. Therefore, this ratio was calculated for 1.0, 2.5, 5.0, 7.5, 10.0, 12.5, and 15.0 ,ug of added T, per 100 ml. The identical percentage of T, was converted to T, at each concentration, averaging 1.69 f 0.05% through ten separate analyses. The constancy of this ratio was determined with each set of 40 unknowns (5 yg of T, was added per 100 ml of hypothyroid serum). 4. T, Recovery Experiments. Exogenous T, was added in nanogram amounts to hypothyroid serum and the resulting mixture measured. In four experiments, recoveries were 93.4, 99.2, 99.5, and 101.4% .with the addition of 5, 11, 20, and 27 ng of T, per ml serum for a mean recovery of 98.6% (Fig. 3). 5. Effect of Added T,. T,, purified as described above and shown to contain less than 0.02% T,, was employed to determine if variations in serum T, would effect measured T,. The validity of using an empirically determined conversion factor in correcting for known methodological conversion of Tq to T, was assessed by adding T, to a hypothyroid and a normal serum. With appropriate correction, the measured T, value remained constant (Fig. 4).

SERUM

p

T3 AND

20 I-

441

GLC

i

NANOGRAMS

line

T4 RY

FIG. 3. T, recovery experiment. The of equivalence. Recovery of added

Clinical

OF T3 ADDED

solid line drawn T, was virtually

at 45’ is the quantitative.

theoretical

Results

The total serum T, concentration was measured in several clinical states. In normal subjects, ranging in age from 20 to 60 years, the mean serum T, was 137 ng/lOO ml (ng 70) with a standard deviation of 23 ng %. Normal male and female subjects had comparable levels of circulating T, and for this reason measurement in both sexes have been combined into a single range.

250

200 E 8 ;: 150 P +* 100

50

FIG. 4. Effect of ing amounts to a hatched bars) and hatched bars). The

added T,. Ta, shown in the solid black bars was added in increaahypothyroid serum containing 60 ng of T, per 100 ml (vertical a hyperthyroid serum containing ZOO ng of T, per 100 ml (diagonal addition did IJQ~ alter the measured T, level.

442

NIHEI

ET AL.

Hypothyroid subjects had a mean serum T, of 67 ng % with a standard deviation of 14 ng %. T, values in this group were about one-half that found in the normal subjects with no overlap between the two groups. There was no significant difference between the patients with primary hypothyroidism and those who had hypopituitarism (3 of the 24). The mean serum T, concentration in the hyperthyroid group with toxic diffuse goiter was 510 ng % with a standard deviation of 131 ng %, There again was no overlap with the normal group and the distribut,ion was skewed toward the lower values in the range (Table 1). TABLE

1

Serum T3 Measurements in Man Subject

No. of subjects

Ta (ng %, mean f S.D.)

Normal Hypothyroid Hyperthyroid

24 24 24

137 f 23 68 f 12 510 f 131

DISCUSSION Gas chromatographic analysis of iodothyronines has been attempted (l-36-9)) but reproducible measurements were obtained only with dilutions of the test solution of 1000 or greater so as to decrease interference by contaminants. These substances, which, in part, are formed during the derivatization reactions, interfere in a nonspecific, nonlinear fashion with electron capture detection and quantitation. Therefore, nanogram quantities of iodothyronines cannot be analyzed unless the derivatives undergo a preliminary purification. This is accomplished with an anionexchange resin and permits measurement of picogram amounts of the iodothyronines. A sensitivity of the order of 50 pg for T, and 100 pg for T, is achieved, allowing for their quantitation in 1 ml of human serum. Slow temperature programming, with an initial 12 min isothermal hold, further purifies the derivatized extracts and permits accurate and reproducible serum determinations. With temperature programming, no inhibition of detector response is observed when extracts containing exogenous T, and T, are measured. Such inhibition, probably attributable to contaminants in the extracts, has been observed when analysis was attempted with isothermal techniques. Despite the known temperature dependence of the response of the electron capture detector, its use in conjunction with slow temperature programming for measurement of T, and T, has been validated by the virtually quantitative recovery of added T,.

SERUM

T3 AND

T4 BY GLC

443

An attempt was made to employ diiodothyronine (T,) and 3,3’,5’triiodothyronine (RT,) for internal standardization. Unfortunately, it was found that the detector responded disproportionately to varying amounts of each iodothyronine. This precluded the use of related compounds as internal standards for quantitative studies and prompted us to resort to external standardization. The external standard, purified derivatives of T, and T, dissolved in benzene containing 1 ng of T, and 2 ng of T, per 5 ~1 aliquot, was injected onto the chromatograph immediately following each sample analysis. Conditions of temperature, gas flow, and detector attenuation were identical to those employed for the serum sample. Prolonged periods of column stability were frequently observed, but significant changes occasionally and unpredictably occurred after a single injection, necessitating retention of the external standardization procedure. The standard curves employed for quantitation of T, and T, in serum were constructed by the addition of pure standards of the iodothyronines to hypothyroid serum at the outset of the procedure. Of note is the fact that determinations on pure standards in solution, i.e., without serum, were dist.ributed on virtually the identical curves. Appropriate corrections were made for yield and for iodothyronines in the hypothyroid serum. Published determinations of the level of T, in human serum have varied widely. Initial estimates by GLC by Hollander (12,13) gave a spuriously high value because of a faulty yield calculation and a failure to account for methodological conversion of T, to T,. Alternative procedures have been proposed utilizing competitive protein-binding techniques (19,21). The values initially reported using this method are higher t,han those we have obtained. Analysis of the competitive binding procedure suggests that the values determined are falsely high because of failure of these workers to account for methoclological conversion (deiodination) of a small proportion of T, to T, during the extraction procedure and for the presence of trace amounts of T, in the T, fraction (22). Although both the per cent of T, converted to T:, and the contamination of T, in the T, fraction constitute a very small proportion of the T, present, they add a significant amount (50-100% of the actual value) to the total T,. This is so since the T:, concentration in serum is approximately one-sixtieth that of T,. Values comparable to those reported here have been obtained by ot,her workers utilizing competitive binding methods with adequate separation of T, and T, and correction for T, to T, conversion (15,22,26,27). A number of workers, utilizing radioimmunoassay techniques, have found the concentration of T, in normal serum to he in the range we have

444

NIHEI

ET

AL.

reported. We recently developed a radioimmunoassay ment of TS in unextracted serum and also find similar 31).

for the measureT, levels (23, 2%

CONCLUSION

A gas chromatographic method has been developed for the measurement of TS and T, in human serum. The ability to simultaneously analyze both hormones should greatly facilitate future studies designed to elucidate the relative importance of these hormones in various physiological and pathological states in man. ACKNOWLEDGMENTS The authors wish to thank Dr. Seymour Reichlin for his helpful guidance and encouragement, and Drs. Saul J. Farber, Stanley B. Troup, and Lawrence E. Young for their advice and support. They also express their appreciation for the skillful technical assistance of Misses Eileen Lesko, Nancy Yule, Colette Thaw, and Harriet Nadel, Mrs. Sandy Ambruster, and Msrs. William Rector, Charles H. Stringham, John Colucci, and Rajendra Gadhok during the various aspects of this study. They also thank Miss Kathy Leslie and Mrs. Helen Boehm for assistance in the preparation of this manuscript. Finally, they would like to acknowledge the helpful technical assistance of Msrs. Bud Boucher, Ken Mohler, and Troy Todd of Tracer, Inc. (Micro-Tek Instrument Division), and Dr. Milton Yates, formerly of Micro-Tek. This study was supported in part by USPHS research grants 1ROl AM 16670-01,2, ROl AM 12866-61, and 2ROl AM 14314-62 (CS’H) and USPHS GRSG grant RR05399-69 (CSH and Summer Fellowship, MCG), as well as a grant from Warner-Lambert Research Labora.tories. Some of these studies were performed in the clinical research units of the University of Rochester School of Medicine (USPHS grant FRd.4) and the New York University School of Medicine (USPHS grant FR-96). REFERENCES 1. STOUFFER, 2. 3. 4. 5. 6. 7.

J. E.. JAAKONMAKI,

P. I., AND WENGER, T. J., Biochim. Biophvs. Acta 127, 261 (1966). JAAKONMAKI, P. I., AND STOUFFER, J. E., J. Gas Chromatogr. 6, 303 (1967). RICHARDS, A. H., AND MASON, W. B., Anal. Chem. 38, 1751 (1966). LOVELOCK, J. E., Nature 189, 729 (1961). BRO(IKS, C. J. W., AND ZABKIEWICZ, J. A., Chapter 3: Gas Chromatography, in “Hormones in Blood,” (C.H. Gray and A. L. Bacharach, eds.), Vol. 2, p. 54. Academic Press, London/New York, 1967. ALEXANDER, N. M., AND SCHEIG, R., Anal. Biochem. 22, 187 (1968). SHAHROKHI, F., AND GEHRKE, C. W., Anal. Biochem. 24, 280 (1968). FUNAKDSHI, K., AND CAHNMAN, H. J., Anal. Biochem. 27, 150 (1969). VOLPERT, E. M., KUNDU, N., AND DAWIDZIK, J. B., J. Chromatogr. 50, 507 (1970).

8. 9. 10. GROSS, J., AND PITT-RIVERS, 11. ROBBINS, J., AND &LL, J.

R., Lancet

262,

439

(1952).

E., The Iodine-containing Hormones, in “Hormones in Blood” (C. H. Gray and A. L. Bacharach, eds.), Vol. 1, p. 383. Academic Press, London/New York, 1967.

452

NAKAO

5 F 2

o1

ARIGA

I

0.4

I

0.8

I

1.2

CONCENTRATION OF ALDEHYDE ( pmole/ml 1

FIG. 2. Substrate

specificity of aldehyde dehydrogenase: (0) propionaldehyde, (0) butyraldehyde. Reaction mixture and conditions as indicated in “Reagents and Materials” except for use of indicated aldehydes as substrate at indicated concentrations. Activity plotted by subtracting control value, obtained by incubating mixture without substrate, from value with the complete system.

(A) acetaldehyde,

liver (7)) in the case of which acetaldehyde showed the highest activity, propionaldehyde exhibited highest activity and formaldehyde no detectable activity. This result was compatible with the one obtained by the conventional spectrophotometric method based on NADH increase. From these results, the methods presented were proved useful in the determination of small amounts of carbony compounds in bioIogica1 materials and, accordingly, in the assay of enzymes that catalyze the formation and decrease of carbonyl compounds. The methods are expected to be applicable to the determination of other carbonyl compounds in more broad categories. However, the present methods were found inapplicable for carbonyl compounds, the hydrazones of which are extremely labile in alkaline solution, such as cinnamic aldehyde and fenchone. SUMMARY

Two 2,4-dinitrophenylhydrazine methods are presented for the determination of small amounts of carbonyl compounds when present in biological materials as a single carbonyl compound. When the hydrazones of the compounds are soluble in ethanolic alkaline solution, a direct method