A Vitamin D3 Steroid Hormone in the Calcinogenic Grass Trisetum flavescens W. A. Rambeck, H. Weiser, and H. Zucker Institute of Physiology, Physiological Chemistry and Nutrition Physiology, Faculty of Veterinary Medicine, University of Munich, Germany, and Department of Vitamin and Nutrition Research, F. Hoffmann-La Roche & Co. Ltd., Basle, Switzerland Z. Naturforsch. 42c, 430—434 (1987); received September 10, 1986 Dedicated to Professor Helmut Simon on the occasion of his 60th birthday Trisetum flavescens, Calcinosis, 1,25-Dihydroxyvitamin D3, Vitamin D 3 Glucosides The grass Trisetum flavescens (golden oat grass, Goldhafer) causes soft tissue calcification in cattle and in sheep. The calcinogenic principle of the plant is the active vitamin D steroid hormone 1,25-Dihydroxyvitamin D3, the major physiological regulator of calcium homeostasis in higher animals. From comparison with synthetic vitamin D metabolites in different bioassays, it is concluded that T. flavescens contains the 25-glucoside of l,25(OH)2D3. This compound, or rather the l,25(OH)2D3 liberated by ruminal fluid, is the calcinogenic factor of the grass. Introduction iological range [9]. Most of these symptoms are Grazing cattle and other herbivores in the Alpine known from hypervitaminosis D and it was therefore region of Germany, Austria and Switzerland develop not surprising when a strong antirachitic activity of a disease called enzootic calcinosis [1—3]. The symp­ the plant was demonstrated [10—12], toms and signs in the affected animals are extensive From a diethyl ether extract of Trisetum flavescens soft tissue calcification, especially of the cardiovascu­ we isolated a fraction showing a strong vitamin D- lar system, kidney, lungs, tendons and ligaments. like activity in several bioassays. A gas chromato­ The animals lose weight, their forelimbs become graphic—mass spectrometric analysis of the purified stiff, their backs are arching and the milk yield is extract revealed that the calcinogenic plant actually reduced [4]. The disease can be of considerable contains vitamin D3, the “animal vitamin D” [13], at economic importance. A similar calcinosis of grazing a concentration of about 0.1 ppm [14], In analogy to animals occurring in Argentina and in Brazil is the formation of the vitamin in the skin of verte­ caused by the ingestion of Solanum malacoxylon [5]. brates, vitamin D3 in T. flavescens is synthesized only However, since this Solanaceae is unknown in under the influence of UV light [15], Part of the Europe, the aetiology must be different. vitamin D3 is present as an ester, but neither the Trisetum flavescens (golden oat grass, Goldhafer, vitamin itself nor the esterified form would be cal­ avoine jaunatre) a common Graminae growing on cinogenic at this concentration [16, 17], Alpine pastures above 500 m, was identified by Peterlik et al. [18] were the first to report a vitamin Dirksen et al. [6, 7] to be the cause of the enzootic D-like substance in Trisetum flavescens that requires calcinosis in the Alpine region. The calcinogenic ac­ no further metabolism in order to exert its effects. tivity is not restricted to grazing animals but can also Active compounds are l,25(OH)2D3, l,25(OH)2D2, be induced experimentally in rabbits [8]. Physiologi­ la(OH)D3 or structural analogs. Furthermore, evi­ cal studies showed that the toxic plant causes an en­ dence for an aqueous soluble vitamin D-like sub­ hanced absorption of phosphorus and calcium, re­ stance in the calcinogenic grass was provided [19, sulting in a considerable hyperphosphatemia and in 20]. These findings and our results from everted gut serum calcium levels in the upper part of the phys- sac transport studies in rats indicated the presence of a substance able to mimick the action of l,25(OH)2D3 [21]. The aim of this study was to find out which la- Reprint requests to W. A. Rambeck. hydroxylated vitamin D metabolite occurs in Trise­ Verlag der Zeitschrift für Naturforschung, D-7400 Tübingen tum flavescens and whether it is a glycosidic-binding 0341 - 0382/87/0400 - 0430 $01.30/0 form that renders the compound water soluble. W. A. Rambeck et al. ■ A Vitamin D3 Steroid Hormone in the Calcinogenic Grass Trisetum flavescens 431 Material and Methods Table I. l,25(OH)2D concentration in rat serum after feed­ ing T. flavescens (residue of diethyl ether extraction). T. flavescens was grown at the “Lehr- und Ver­ suchsgut Schleißheim der Universität München”. Addition per l,25(OH)2D pg/ml serum Vitamin D metabolites and their glycosides were kg of diet (pool from 6 animals) kindly supplied by Dr. W. Meier, Pharmaceutical — (neg. control) 57 Dept., F. Hoffmann-La Roche & Co. Ltd., Basle. 90 g T. flavescens 106 In the prophylactic chick assay, newly hatched 200 g T. flavescens 375 male broiler chicks were put on a vitamin D-deficient diet containing added levels of vitamin D3 or its de­ The increase in rat serum l,25(OH)2D concentra­ rivatives. After three weeks weight gain, serum cal­ tion was not necessarily due to l,25(OH)2D3, since cium and per cent bone ash of phalanges I and II of the competitive protein-binding assay used responds the middle toes were determined. to l,25(OH)2D3 as well as to l,25(OH)2D2. The same In the curative assay the chicks were fed a vitamin is true for our finding that a water extract of D-deficient diet for ten days. During the following T. flavescens was able to replace radioactive experimental period vitamin D3 or its derivatives l,25(OH)2D3 from its intestinal receptor and there­ were added to the diet. After six days the animals fore contained l,25(OH)2D. were killed and serum calcium and duodenal cal- In order to find out which vitamin D metabolite is cium-binding protein (CaBP) were determined. present in this plant, we compared the bioactivity of For the curative Japanese quail assay newly l,25(OH)2D3, l,25(OH)2D2 and T. flavescens in dif­ hatched animals were put on a vitamin D-deficient ferent bioassays. While in rats the two vitamin D diet for ten days. After this period, a diet containing metabolites and the extract of the grass behaved very vitamin D or vitamin D derivatives was fed for five similarly [26, 27], there was a fundamental difference consecutive days. Weight gain, serum calcium and in rachitic chicken. The synthesis of CaBP, which is a bone ash per cent were determined. good indicator of the molecular expression of the The curative X-ray test in rats was performed as hormonal action of vitamin D metabolites, is shown described by Weiser [22], The degree of healing was in Table II. assessed by scoring values. Compounds were given From these and other results [27] it can be con­ per stomach tube in propylene glycol/ethanol 10:1. cluded that l,25(OH)2D2 is about 10 times less active CaBP was determined by the ion exchange proce­ in chicken than l,25(OH)2D3, while the calcinogenic dure according to Wasserman et al. [23]. CaBP is compound in T. flavescens shows a similar antirachi­ expressed as the percentage of radioactivity in the tic activity in rats and in chickens. Thus all biological supernatant from total radioactivity per mg of evidence indicates that the active principle is protein. l,25(OH)2D3 and not l,25(OH)2D2. Determination of l,25(OH)2D in serum and the The apparent paradox that the vitamin D metabo­ assessment of the binding capacity of T. flavescens lite in T. flavescens is water soluble can be explained extract to the intestinal l,25(OH)2D3 receptor was carried out by the competitive protein-binding assay Table II. Synthesis of duodenal CaBP in rachitic chicks. according to Mallon et al. [24], Curative assay. Mean values ± S.D. (n = 10 per group). Addition in nmol CaBP kg of diet Results and Discussion — (neg. control) 1.24 ± 0.33 While vitamin D3 in T. flavescens was found in the 1.3 l,25(OH),D3 2.46 ± 0.63 ether extract of the plant, the vitamin D metabolite­ 2.6 l,25(OH),D, 4.17 ± 1.24 like compound occurred in the residue of the ether 5.2 l,25(OH)2D3 5.06 ± 1.25 extraction or in the aqueous extract. When this frac­ 10.4 l,25(OH),D, 3.09 ± 1.14 tion of the calcinogenic grass was fed to rats, 20.8 1,25(OH)iD2 4.01 ± 1.13 41.6 l,25(OH)2D2 6.09 ± 1.89 l,25(OH)2D concentration in serum increased (Table I). Similar results were obtained when T. 200 g T. flavescens ether extract residue 4.28 ± 1.18 flavescens was fed to cows [25]. 432 W. A. Rambeck et al. • A Vitamin D, Steroid Hormone in the Calcinogenic Grass Trisetum flavescens by the finding that l,25(OH)2D3 in T. flavescens is of T. flavescens with two la(O H )D 3 glucosides and present as a glucoside. Treatment with glucosidases with the three possible l,25(OH)2D3 glucosides. In or with ruminal fluid releases the free steroid Fig. 1, the calcium excretion via the egg shell of hormone [28]. In Solanum malacoxylon, the cal­ Japanese quails is shown. At the concentration cinogenic plant of South America, also a glycoside of administered, neither la(O H )D 3-3-glucoside nor the l,25(OH)2D3 is the active principle, but neither the disaccharide la(OH)D3-3-cellobioside increased Ca nature of the carbohydrate moiety nor the connec­ excretion via the egg shell. In a curative X-ray test in tion to the aglycone have been elucidated.