Saturated hydrocarbons in human femoral arterial tissues and plaques

ARCHIVES

OF

BIOCHEMISTRY

AND

BIOPHYSICS

Letters Saturated

Hydrocarbons Arterial

Tissues

in Human and

113,

245-252

to the

(1966)

Editors

The residues were redissolved in CC14 and their infrared spectra were determined in a PerkinElmer model 137 infrared spectrometer. The residues were again evaporated and redissolved in n-hexane, and their ultraviolet spectra were determined in a Beckman DK-2A spectrometer. The hexane solutions were next chromatographically fractionated on alumina columns (prewashed with n-hexane rmtil no infrared and ultraviolet absorpt,ion effects were noticeable in the washing solutions). The adsorbant to sample ratios were at least 3OOO:l. The columns were eluted with 100 ml of each of the four conseclltive eluents: n-hexane, carbon tetrachloride, benzene, and methyl alcohol. Each of the four eluates of the samples was analyzed by infrared and ultraviolet spectroscopy and by thin-layer chromat,ography. The thin layers consisted of Silica Gel G and were prewashed with chloroform to remove any hydrocarbon impurities that might be present, and then developed with n-hexane and in different series of runs with n-hexane-ether-acetic acid (70:30:1, v/v/v; and 94:1:5, v/v/v, respectively). The thin-layer chromatograms were first examined under ultraviolet light to detect naturally fluorescent compounds such as aromatic hydrocarbons. No such components were observed. Next the thin-layers were sprayed with Rhodamine GG visllalizer and photographed under ultraviolet light. This thin-layer chromatographic technique has been described in detail by Murphy et al. (4). All experiments were conducted with freshly dist,illed solvents that had been spectroscopically analyzed and found to be free of saturated hydrocarbon or other impurities. Control experiments showed that the solvents contained less than 0.4 mg/lOO ml residue. No stopcock grease was employed on the developing tanks or in the laboratory, and the samples did not come in contact with plastic or rltbber implements. All glassware was cleaned prior to use with an 85:15 (v/v) solution of hot, concentrated H&O4 and HNOI. Figures la and lb show (as well as the plates developed with hexane-ether-acetic acid) that both the plaques and the artery wall tissues contained components t,hat had Rf values indentical to the n-alkane standard, rb-octadecane, and other n-alkanes with carbon chains somewhat longer or shorter than n-octadecane. It was concluded that t,hese components were saturat’ed

Femoral

Plaques

Traces of saturated hydrocarbons occur extensively in nature. In a recent report, Nagy et al. (1) reviewed the saturated hydrocarbons found in a variety of plant and animal tissues and described additional plants that contain long-chain n-alkanes. Saturated hydrocarbons apparently were present even in biological matter in the geological past. Oro et al. (2) reported finding branchedchain and n-alkane hydrocarbons of biological origin in sedimentary rocks approximately two billion years old. In spite of their wide occurrence in nature, relatively little is kn0me-n with certainty about their presence in human tissues. For example, the experiments of Nicolaides (3) show that hydrocarbons on the surface of human skin are most likely contaminations. The present investigat,ion was undertaken in an attempt to learn something about the possible presence of saturated hydrocarbons in human tissues with proved, ultramicro analytical methods that have been developed during the past few years. Human femoral arteries in formaldehyde were obtained through the courtesy of Professor Charles T. Dotter, M.D., of the University of Oregon Medical School. The artery samples were first washed for 10 holtrs in distilled water and then dried overnight at 70°C. Xext, the plaques were dissected from the arterial wall and ground to a fine powder. A microscopic examination of the liberat,ed plaque material showed that a good but not quantitative separation had been attained between the plaques and the arterial tissues. The artery wall tissues (consisting mainly of endothelium and the media) separated from the plaques, the ground plaques, and a control sample of 600 ml of formaldehyde’ xere analyzed for saturated hydrocarbons. All samples were extracted at room temperature with a 9:l (v/v) solution of benzene-methanol four times consecutively for a period of 2 hours. The combined extracts were evaporated to dryness under a stream of N? (water pumped and passed through a Matheson molecular sieve gas purifier) at 50°C. 1 The analysis of 600 ml of formaldehyde was probably a rather exacting control because the samples were washed for 10 hours prior to analysis to remove free, unrearted formaldehyde. 245

LETTERS

TO

TI-IJ:

FIG. 1. (a) -&ending thin-layer chromatogram temperatrue and visualized with Rhodamine tions: 1, the urrfractionated berlzene-nlethallol alrunina col~~mn; 3, carbon tetrachloride eluate; ate, and 6 and 7, 25 pg each of n-octadecane The cholest,erol standard remained at the origin lipids appears as a faint spot slightly above veloping solvent. (b) Thill-layer chromatogram graphic eluates, and standards identical to and hydrocarbons of high enough molecular weight to volatilization under the experimental escape conditions applied. The spectroscopic data confirmed these findings. By using various solvent systems it, was also found that the arterial samples and plaques had other components with RI values identical to t,hose of the monounsaturated alkene, n-octadecene, and cholesterol. In addition, there were at least three lmidentified lipid components present. The artery walls examined in this study contained 330 ppm and the plaques, approximately 300 ppm of saturated hydrocarbons. The formaldehyde solution contained only traces (0.1 ppm) of these compounds and, because of quantit,at,ive considerations, would probably not, be the source of the arterial hydrocarbons. Of course it is difficult to be certain that, hydrocarbons were present in the artery samples in vizlo, and admittedly it would be preferable to analyze the tissues immediately after removal. In the present study t,his was not practicable. It also appears unlikely that the hydrocarbons were laboratory contaminations becallse the previously described t,echniques were used, and these techniques have been found adeqrlnte, through repeated experience, in excluding contaminations. The physiological role of the saturated hydrocarbons in the femoral arteries and plaques is not known. There cordd be three possible results of their presence ill the tissues: (a) they are inert,

EUITOl:S

developed with n-hrxane a1 roo’m of arterial plaque extracts. Applicaextract; 2. n-hesane elrlate from 4, benzene eluate; 5, methyl alcohol elrland cholesterol standards, respect,ively. with n-hexane; one of the unidentified the origin in application 2 with t,his deof artery wall extracts, chromatoarranged in the same order as in Fig. la. 6G,

(b) they are not inert and give rise to a bodily response by an unknown mechanism, and (c) t)heg are part of the normal metabolic machinery but of unknown function. However, one cannot dismiss the possibility that the hydrocarbons are products of certain lipids that decomposed at one time after the removal of the artery from the individual. Should this be the origin of the saturated hydrocarbons, their presence in the arterial samples may help t,o elucidate the structure of their parent, lipids. Rouser (5) suggested that hydrocarbons in brain tissues may have had such an origin. On the other hand, Riley et al. (6) found that human liver contains hydrocarbons t,hat were probably digested and carried to the liver tissues by the blood stream. In view of these it may be illuminating to examine arterial samples of individuals not) affected by arteriosclerosis. This investigation may be helpful to point ollt the applicabilit,y of ultramicro hydrocarbon analytical methods to tissue samples and the ubiqllity of saturated hydrocarbons in biological matter. ACKIGOWLEDGMENTS The authors would like to thank Dr. Charles T. Dotter of the Iiniversity of Oregon llledical School for providing the artery samples; Mar>Ellen O’Reilly and Sister N.T.J. Murphy of the Universit’y of California, San Diego, for their assistance and their suggestions dllring the course

LETTERS

TO

of this study; Prof. H. C. Urey for his interest in this work, and Dr. Paul B. Hamilton of the A. I. duPont Institute of the Nemours Foundation for critically reading the manuscript. The research was supported by NASA research grant NsG-541.

THE

247

EDITORS TABLE

INTRINSIC

VIEXOSITIES

OF GELATIN

OF POLYAMINO

IRRADIATED

Polyamino

I IN

FRANCO

GAZZARRINI*

BARTHOLOMEW

Department University Received Radiation

NAGY

of Chemistry of California, San Diego September IS, 1966 Degradation the

of Solid

Polyamino

Acids

[?I”

Gamma irradiation of solid polyamino acids in vacua (with a Cobalt 60 source) (2, 3), and viscosity and osmotic pressure measurements have been described (4). Viscosity measurements were taken for each polyamino acid at concentrations of 1.5, 1.2, and 0.9% after exposure to 40 Mrads. Osmotic pressure measurements were made at concentrations between 0.20 and 0.057, after exposure to the same dose. At these doses of irradiation we could not detect any amino acid transformations similar to those reported for polyamino acids irradiated in aqueous medium (5)) and there were no alterations Montecatini,

Novara,

Poly-L-glutamic.Na in 0.1 N borate buffer, pH 11.5 Poly-L-proline in trifluoroethanol Poly-L-valine in trifluoroacetic acid Poly-L-glycine in trifluoroacetic acid Poly-L-leucine in trifluoroacetic acid Poly-L-alanine in trifluoroacetic acid Poly-L-lysine HCl in HnO Glum-LysB-TyIg in H20b Gelatin in H*O (calfskin)

Control

40 Mrads

1.875

0.350

1.140

1.125

0.256

0.196

0.286

0.265

2.580

0.625

1.050

0.617

2.900 1.075 0.435

0.435 0.437 0.335

in

State

METHODS

address:

AND

STATE

0 171 expressed in deciliters/gram. b Exposed to 25 Mrads and not to 40 Mrads. c Exposed to 15.5 Mrads and not to 40 Mrads.

Formation of long-lived peptide radicals (-CONHeR-) occurs upon gamma irradiation of peptides in an evacuated solid state (1). The direct cleavage of the C-H bond of the peptide chain represents but one path for the production of the long-lived radicals. These stable radical sites are also introduced through hydrogen abstraction by reactive species formed through the cleavage of N-C and C-C linkages of the peptide chain. The number of such broken bonds should be reflected by a decrease in viscosity as well as in molecular weight. The study reported here was undertaken to test this prediction.

* Present

DRY

acid

REFERENCES 1. NAGY, B., MODZELESKI, V., AND MURPHY, M. T. J., Phytochemistry 4, (1965). 2. ORO, J., NOONER, D. W., ZLATKIS, A., WIKSTROM, S. A., AND BARGHOORN, E. S., Science 148, 77 (1965). 3. NICOLAIDES, N., Personal communication. 4. MURPHY, M. T. J., NAGY, B., ROUSER, G., AND KRITCHEVSKY, G., J. Am. Oil Chem. Sot. 42, 475 (1965). 5. ROUSER, G , Personal communication. 6. RILEY, R. F., HOKAMA, Y., AND KRATZ, P., Cancer Res. 18, 825 (1958).

ACIDS

THE

Italy.

in the infrared patterns of these irradiated compounds in KBr disc form. After the seal of an irradiated sample was broken in open air, the compound was added to the solvent of choice. No attempt was made to free a solvent from oxygen. Thus the procedure used here is identical with that employed by us in previously reported investigations. RESULTS

AND

DISCUSSION

From the experimental data (Tables I and II) it appears that all polyamino acids, as well as gelatin, irradiated in an evacuated solid state indeed have lower intrinsic viscosities and lower number average molecular weights as the result of the molecular degradation. This observation is in line with data reported for soluble Jibrous proteins. For instance, upon gamma irradiation in the solid state at 2.5 Mrads, bovine fibrinogen exhibits a decrease in weight-average molecular weight from 398,000 to 62,000 and a decline in intrinsic viscosity. There occurs also a lowering in solubility suggesting that besides fragmentation some aggregation of the protein molecules does take place (6). In the case of insoluble fibrous proteins one observes increased solubility after irradiation due to the molecular breakdown (7). It should be emphasized that the lower viscosities and molecular weights as well as t,he number of broken bonds (PO) and yield of fractures per 100