A NOTE ON THE BIOLOGICAL ASSAY OF VITA- MIN D IN FISH LIVER OILS : G. KARMAKAR & M. N. RAO. (P. H. S.) PAGE 223 AA specimenspecimen photographphotograph (X(X 6)6) ofof thethe stainedstained bonesbones ofof thethe kneeknee jointjoint ofof aa rachiticrachitic ratrat (line(line testtest valuevalue 1.0).1.0). April, 1947] VITAMIN D IN FISH LIVER OILS : KARMAKAR & RAO 223 ' A NOTE ON THE BIOLOGICAL ASSAY on rachitic diet?a prophylactic test \ For both the tests, colonies of rats reared OF VITAMIN D IN FISH LIVER OILS specifically lip with low vitamin D reserves are recom- By G. KARMAKAR mended. But our experience in Calcutta showed a and difficulty in rearing up large specific stock of young rats for the purpose under the tropical N. RAO M. climate. The other alternative for us was to and {Section of Biochemistry, Nutrition Physiological use a colony of rats on stock diet reared up in and Public Hygiene, All-India Institute of Hygiene darkness. When these colonies of rats Health, Calcutta) partial were being used it was often found that the Introduction degree of extreme rickets that is wanted in the The biological methods commonly em- curative or line test did not satisfactorily deve- for of vitamin D are the Moreover, the values of the line test in 'ployed estimation lop. line test' and the ' ash test \ In the former, the same group of experimental animals varied the degree of calcification of bones produced in to a considerable extent. The values of the heal- a rachitic rat by the test substance is taken ing in the line test are liable to variation not as an index of vitamin D i.e. a because of the biological material but also ' its content, only curative testIn the latter, the potency of due to personal error in quantitative assessment the test substance is taken as proportional to of healing. We therefore wanted to try with the degree of prevention of rickets in animals the stock of available rats the efficacy of the Table I Line test A B D E G Number of ir, -p, Experimental number of Average imii D of Serial Degree of period : rats drops VltaT7TnI.U. ^dose **??** of - oil* days days dSL|f Standard oil 4.0 24 9.3 0.97 29 10 3.5 3.66 3.5 l 3.0 12 4.65 0.67 29 10 3.0 4.0 2.92 2.5 2.5 2.5 0.78 1.89 29 10 10 0.25 11 0.25 12 0.25 13 0.25 14 0.25 0.55 15 0.25 16 | 1.00 17 1.00 18 I 1.50 Shark liver oil 29 10 19 2.25 24 Unknown 1.88 20 1.5 12 Unknown 29 10 21 0.75 22 0.75 0.88 23 1.0 24 1.0 M/nr oil nwke 1 oc'and*? ef|i'ivaie"t to ?2 i.u. tm&beeSclficS.the "e8rce 0f heali"K in * s"le '? ?'hiA ? *,? ">.ket8 and 6 indices 224 THE INDIAN MEDICAL GAZETTE [April, 1947 alternative ash test in which the accurate per- Subrahmanyan, Duckworth and Godden (1939). centage of ash is taken as the criterion instead The method consists in removing mechanically of the healing values of line test wherein the as much of the soft parts from the skeleton as personal error is likely to vitiate the results. possible and subsequently incubating the skele- ton with papain to digest the remnants. By Experimental this method it was found that even the inter- The diet used us vertebral ligaments are digested and the ver- following rachitogenic by ' was a high calcium and low phosphorous diet :? tebrae are loose. The short bones, the vertebrae, the small Rachitogenic diet bones and the long bones of each of the animals Per cent are preserved separately for ashing to find any a Yellow maize (powder) .. 60 relative differences. The ashing to constant Whole wheat .. .. 25 weight is done after extracting the bones in .. .. 10 Gelatine alcohol and ether for 6 hours to remove the Calcium carbonate .. .. .. 4 last traces of fat. The results of the Sodium chloride .. .. .. 1 analysis are given in table III. Young rats weighing about 30 gm. were kept on the above diet for 4 weeks in complete Table III darkness. During this preparatory period the rats were weighed twice a week and also the Ash test amount of food consumed daily was weighed to detect Total dose of vitamin D any loss of appetite which is supposed ash the whole Average percentage to lead to spontaneous healing of rickets. After given during (of the entire skeleton) making sure that all the animals developed experimental period complete rickets by doing a line test on some of them the rats were divided into five groups : Standard oil three groups on graded doses of standard oil and the fourth and fifth groups on oil of unknown 30 drops 59.06 (11.6 I.U.) potency. The test period was for 10 days and oil was are the given in 2 doses. The results 60 drops 59.40 given in table I. (23.2 I.U.) Values of average healing (column H) are plotted against the logarithm of the doses Shark liver oil (column C) and a mean curve is drawn through them. 60 drops 59.09 (5 I.U. as found line test) From this curve the abscissae are found out by corresponding to the average healing for the 120 drops 59.26 shark liver oil, and the values are calculated (10 I.U. as found by line test) in table II. Table II Conclusions Table III shows that of the Antilog of irrespective graded the abscissae doses of vitamin D the percentage ash is uni- Dose in Vitamin D constant that there has been Average Abscissae (represents formly signifying drops healing the (I.U./c.c.) spontaneous of rickets. the D healing Probably, vitamin time that is taken for the ash i.e. content) greater test, nearly 8 to 10 weeks, was responsible for the spontaneous healing. But in the case of the 24 1.88 0.32 2.1 15.8 line test the shorter time gives lesser chances of 12 0.88 1.97 0.93 14.0 spontaneous healing. Hence the line test appears to be the test of choice whenever the of rats cannot be reared Starting with this oil of known potency an specific stock colony ash test was done on a group of 22 rats obtained up for any reason. The line test is used as a partly from the laboratory stock and partly routine in this laboratory. from the market. The rats were divided into The method of reporting the results with line test from this be of interest. four groups, two for feeding the standard oil laboratory may and two for the shark liver oil and the doses The values allotted to the degree of healing on To were given from the beginning of the experi- depend purely personal judgment. ment. The doses selected were within the limit avoid this personal error the ends of the bones of prophylactic doses, i.e. one-fifth of the curative are all enlarged and photographed. This facil- dose and even less and therefore one would itates not only a permanent record but of expect partial calcification of the skeleton. The check up by different workers. The specimen on a flat white feeding was continued for about 7 weeks after is mounted surface and illumi- which the animals were killed and the skeleton nated with two 50 watt Philips photographic separated from the rats by the method of lamps at such angles (about 45?) as to avoid April, 1947] FIFTY YEARS AGO 225 any dark shadows. The camera (Contax II with Sonnar 1.5 lens) is mounted on a universal stand and Zeiss micro-enlarger X 2 used for magnification. A sample photograph is an- nexed (see plate X). In this connection it may be mentioned that during our estimation of vitamin D content of different food samples by the line test we could not produce complete rickets on rats purchased from the market (weighing 25 to 30 gm.) when we kept them on the rachitogenic diet for ?ven 6 weeks or more. This shows that unless 'lie animals are bred in the laboratory on a res- tricted diet it is useless to attempt the line tost method for the estimation of vitamin D. I he ash test mentioned on the other hand is in our experience a failure in a hot climate although the animals are bred in the laboratory. Summary The rearing up of a specific stock colony for vitamin D assay in a tropical place like Calcutta is difficult. When the stock is not Quite satisfactory ash test is not of much value. The line test is the test of choice. Acknowledgment The work was undertaken under the direc- tion of Dr. G. -Sankaran, Professor of Biochemistry, Nutrition and Physiological Hygiene. We sincerely thank him for his co- operation and guidance. REFERENCE Sttbrahmanyan, V., Duck- Biochem. ./., 33, 1421. worth, J., and God den, W. (1939). .