Amygdalin (Laetrile) and Prunasin
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Proc. NatL Acad. Sci. USA Vol. 78, No. 10, pp. 6513-6516, October 1981 Medical Sciences Amygdalin (Laetrile) and prunasin (3-glucosidases: Distribution in germ-free rat and in human tumor tissue [cyanogenic glucosides/neutral I3(1- 6)- and (1-4)-glucosidases/gentiobiose] JONATHAN NEWMARK*, ROSCOE 0. BRADY*, PHILIP M. GRIMLEYt, ANDREW E. GAL*, STEPHEN G. WALLER*, AND J. RICHARD THISTLETHWAITEO *Developmental and Metabolic Neurology Branch, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20205; tDepartment of Pathology, Suburban Hospital, Bethesda, Maryland 20814; tUniformed Services University ofthe Health Sciences, Bethesda, Maryland 20814; and Department of Surgery, George Washington University Medical Center, Washington, D.C. 20037 Contributed by Roscoe 0. Brady, July 20, 1981 ABSTRACT Amygdalin, the gentiobioside derivative of man- enzyme was unable to hydrolyze gentiobiose, the disaccharide delonitrile commonly referred to as Laetrile, is presently under component ofamygdalin, suggesting a requirement for an aryl intensive investigation as a potential cancer chemotherapeutic or alkyl aglycone residue for enzymatic activity. The natural agent. Because of this interest, we investigated the activity of (3- occurrence ofthis enzyme was unusual, beingparticularlyplen- glucosidases that cleave glucose from amygdalin and from pru- tiful in feline kidney but also present in rat and rabbit kidney nasin (mandelonitrile monoglucoside) in tissues from germ-free and rodent intestine. It was notably absent in human kidney rats and in normal and neoplastic human tissues. Rat and human preparations. This enzyme and the subsequent activity of a small intestinal mucosa contain high levels of activity of glucosi- (1-4)-glucosidase on the prunasin produced by the ((1-*6)- dases that act on both of these cyanogenic glucosides. Release of glucose from these compounds was not detected in any of the hu- glucosidase were considered to be important in the metabolism man neoplastic tissues examined in the present study. These ob- of exogenous amygdalin because mandelonitrile can sponta- servations are consistent with reports of cyanide toxicity through neously release cyanide in vivo. Thus, it was suggested that, if the oral use of amygdalin or prunasin and pose serious questions amygdalin exerts a tumoricidal effect by release of cyanide, a concerning the alleged tumoricidal effect of amygdalin. tissue susceptible to amygdalin should contain the required (- glucosidases. In order to better characterize the occurrence of Amygdalin (D-mandelonitrile-,-D-glucosido-6-,B-D-glucoside) amygdalin and prunasin glucosidases, we have extended the has been promoted (1) and, in 23 American states, legalized as previous study (13) to an examination oftissues from germ-free an oral chemotherapeutic agent for "terminal" cancer. These rats and normal and neoplastic human tissues. endeavors were endorsed despite documented heterogeneity ofamygdalin and "Laetrile" preparations (2, 3), a dearth ofevi- dence of the effectiveness of amygdalin on animal (4-6) and MATERIALS AND METHODS human neoplasms (7, 8), and serious questions regarding po- tential cyanide toxicity. Amygdalin was obtained from Aldrich. Prunasin was prepared Schmidt et al (9) demonstrated that oral administration of as described (13). Gentiobiose was purchased from Sigma. amygdalin in doses equivalent to the recommended human tu- Germ-free male rats (200 g) were obtained from Charles moricidial doses along with the sweet almond preparations con- River Breeding Laboratories, Wilmington, MA. Upon arrival, taining the amygdalin-hydrolyzing enzyme complex emulsion the rats were sacrificed by asphyxiation with solid C02; the tis- produced high levels ofHCN in serum, clinical signs ofcyanide sues were removed and homogenized by hand in 10 vol ofcold toxicity, and death of 6 of 10 experimental animals. Khandekar distilled water. The mixtures were centrifuged at 500 X g for and Edelman (10) administered amygdalin to rats intraperito- 10 min, and the supernatant suspensions were assayed for en- neally and demonstrated dose-dependent mortality and clinical zyme activity. The quantity of glucose enzymatically released evidence of cyanide toxicity without the concurrent use of al- from amygdalin, prunasin, and gentiobiose was determined mond 13-glucosidases. However, Ames et at (11) found that par- with glucose oxidase reagent (Sigma) as described (13). 3Glu- enteral administration ofamygdalin resulted in excretion ofthis cosidase activity was also measured with 4-methylumbelliferyl- compound in the urine primarily in an unchanged form. In hu- ,B-D-glycopyranoside (Sigma) as substrate at pH 4.5 in acetate mans, Moertel et at (12) found that intravenous infusion of buffer and at pH 7.5 in phosphate buffer (14). Glucocerebros- amygdalin produced neither cyanidemia nor signs oftoxicity but idase activity was also determined in the tissue specimens by that oral administration resulted in significant blood cyanide using ['4C]glucocerebroside as described (15). Protein was de- levels and that, in one case, oral amygdalin plus almond extract termined by the procedure of Lowry et at (16). produced transient symptoms of cyanide intoxiciation and fur- Human tissues from 19 patients were obtained at operation ther increase ofblood cyanide. These studies seemed to indicate from the Department of Pathology, Suburban Hospital, Be- that amygdalin was probably metabolized in some portion(s) of thesda, MD. Patients were unselected. The tissues were re- the gastrointestinal tract. frigerated until homogenization 2-16 hr postoperatively. Tis- Freese et at (13) isolated and partially purified a neutral sues were processed in the same manner as before, and the (3(1-*6)-glucosidase that catalyzes the hydrolysis ofthe terminal tissue diagnosis on each specimen was subsequently confirmed glucose moiety of amygdalin and clearly differentiated this en- microscopically. Two specimens ofhuman small intestine were zyme from previously known mammalian /B-glucosidases. The obtained at autopsy. Postmortem diagnosis for each patient was cardiac disease, and no gross pathology was seen in the abdo- The publication costs ofthis article were defrayed in part by page charge men. The autopsies were performed the day after death, and payment. This article must therefore be hereby marked "advertise- the bodies had been refrigerated for an unstated time before ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. autopsy. 6513 Downloaded by guest on October 3, 2021 6514 Medical Sciences: Newmark et aL Proc. Nad Acad. Sci. USA 78 (1981) RESULTS cessing ofthe samples, or both. Evidence that the latter was at least partially responsible was found by allowing aliquots ofthe Enzymes that catalyze the cleavage of glucose from amygdalin two most active samples in 1:1 dilution with 5.6 mM sodium and prunasin were present in the small intestine and intestinal azide to stand at room temperature for 24 hr before assaying; contents of germ-free rats and in the kidney (Table 1). These this caused a 33% reduction in amygdalin-hydrolyzing activity (3glucosidases were practically absent in the stomach and in the and a 50% reduction in prunasin-hydrolyzing activity. large intestine. Both proximal and distal halves of the small in- testine mucosa contained both amygdalin- and prunasin-cleav- DISCUSSION ing activity. The colon contents showed significant activity The biological roles of mammalian (3glucosidases that hydro- "downstream" from the site of high small intestinal mucosal lyze amygdalin and prunasin are not understood. The high level activity. This observation is consistent with shedding ofmucosal of 3(1-*6)-glucosidase activity in kidney from several species elements or secretion ofthese enzymes, or both, into the lumen (but not human renal tissue) (13) may be related to the ne- of the small intestine. The possibility of a bacterial source of phritogenic glycopeptide in rat glomerular basement mem- these enzymes is remote because the rats were germ-free. brane examined by Shibata and Nakanishi that contains the The data obtained with human tissue confirm the localization unusual 8(1--6)-glucosylglucopyranosidic bond (19, 20). Met- of these 13-glucosidases to the small intestine (Table 2). None abolic turnover of this component would presumably require of the human neoplastic tissues examined exhibited either the activity of such a glucosidase. The presence of the poly- amygdalin- or prunasin-hydrolyzing activity. All of the tissues peptide portion of this molecule could satisfy the apparent re- contained significant glucocerebroside-f3glucosidase activity as quirement for an ailcyl or aryl aglycone moiety for the renal well as glucosidase(s) that catalyze the hydrolysis of 4-methyl- (8(1- 6)-glucosidase. Human feces exhibit a wide variety ofgly- umbelliferyl-,&D-glucopyranoside. Catalytic activity with the coside-splitting enzyme activities (21) that are presumed to be latter substrate was much greater at pH 4.5 than at pH 7.5 in largely of bacterial origin. The natural substrates of the intes- all tissues except rat kidney and rat and human small intestine. tinal enzymes that catalyze the hydrolysis ofglucose from amyg- The pH optimum of rodent, kidney 8(1-+6)-glucosidase is 7.5 dalin and prunasin remain to be identified. (13). The comparatively high activity with the fluorogenic sub- Laetrile advocates have argued thatglycosidically substituted strate at this