MICROBIOLOGICAL ASSAY OF

VITAMIN B12 AND ITS ROLE

IN EVALUATION OF

LIVER EXTRACTS \ By S. P. pE

and

B. Ml/KERJI (Central Drugs Laboratory, Government of India,

Calcutta 12)

N ?'

Introduction

Although a widely recognized and important therapeutic preparation, the standardization of liver extracts has offered considerable difficulties to manufacturers and laboratory workers. In

spite of repeated attempts extending over the last 30 years, no satisfactory

'

laboratory method' has so far been considered worthy of general acceptance. The British Pharmacopoeia lays down specifications for the manufacture of

liver extract and only demands that '30 millilitres of the liquid extract of the liver should contain the equivalent of 240 gm. of fresh liver This method naturally does not ensure any check over the specific active principle of liver extract, which is responsible for increasing the number of red corpuscles in the blood of persons suffering from pernicious or other types of anaemia. In the United States, a more

rational method of standardizing the product has been introduced. The Anti-Anaemia Pre-

parations Advisory Board allots a certain '

unitage ' to all liver extracts manufactured in

that country. These units are declared after clinical trials of these products in hospitalized patients suffering from particular types of anaemia. This is undoubtedly a good method for estimating the potency of liver extracts, but obviously a method which demands considerable paraphernalia and special types of hospitals for housing of anaemia patients. This is not eas\' to organize in India and, in fact, in many other countries faced with the problem of liver extracts standardization. A '

laboratory method ' of assay of this preparation will, there- fore, always remain a prime necessity. In an earlier paper from this laboratory,

Ghosh and Mukerji (1950) suggested a modified '

laboratory method' (based on the work of

Muller, 1935) for standardization of liver extracts depending upon the reticulocytic res-

ponse in domestic pigeons. Although this method appears to be promising and gave fairly dependable results, it is a time-consuming pro- cedure and suffered from the inherent defect that the ' test object' (pigeons) must be absolutely free from intercurrent parasitic infections, which is not always easy to ensure. Several other methods have also been recommended from time

to time but have not found favour with the

workers.

The difficulty of formulating a suitable '

laboratory method ' of assay is due to the fact

that so far the actual anti-anaemia factor or

factors in liver extracts have not been clearly defined. Shorb (1948) in the United States dis- covered a fraction from liver extract, which

supported the growth of a strain of Lactobacillus lactis. Rickes et al. (1948) and West (1948) in U.S.A. and Smith (1948) in England isolated this factor from liver extracts and named it

'Vitamin B12 \ They were of the opinion that this vitamin was the active anti-anaemia factor

in such preparations. Spies et al. (1948, 1949) carried out extensive clinical trials and proved beyond doubt that vitamin B12 was the most potent anti-anaemia fraction in liver extracts, in that only an infinitesimal amount of this

principle was required in comparison to folic

acid, thiamine, etc., in bringing about reticulo- cyte response in suitable anaemia cases. Sub-

sequent clinical trials by Hall and Campbell of the Mayo Clinics (1948), and those at the Royal

404 THE INDIAN MEDICAL GAZETTE [Sept., 1951

Victoria' Infirmary, Newcastle-upon-Tyne, England (see Editorial, Lancet, 1949), confirmed the previous clinical findings and established the value of vitamin B12 as one of the most

important erythrocyte maturing factors. Shorb's (loc. cit.) announcement that the

micro-organism, Lactobacillus lactis Dorner, responded to a factor in liver extracts and its identification as vitamin B12 opened up a new field before laboratory workers and indicated, for the first time, that liver extracts could perhaps be more accurately assayed than before by estimating their vitamin B12 contents. This was

supported by the works of Shaw (1949), who estimated the B12 factor in liver extracts, and Cuthbertson et at. (1949), who showed that the clinical response of liver extract in antenna

patients showed a very close relationship to the actual amount of vitamin B12 present in these

preparations. Later, Cuthbertson and Smith

(1949) published in detail a microbiological method for assaying the vitamin B12 content

in liver extract.

The present work was undertaken in this

laboratory as part of its programme for the

screening and control of liver extracts qf indifferent potency, which are frequently offered for sale in the Indian markets. The samples were collected from various sources and can be considered to be representative in character. From the results, which are recorded, it appears that with proper precautions and controls this method of microbiological assay of vitamin B12 in liver extracts places in the hands of laboratory workers a fairly reliable and easy guide in the evaluation of the anti-ansemic potency of these preparations, which had not been possible by previous methods. Some of the items tested by the microbiological method as described later were duplicates of samples already tested by trained workers by the

' clinical methodIn such cases, the results have also been very promising, so much so, that it is gradually becoming clear that the microbiological method of assay may actually give more precise results than ' clinical tests' and that with further

development of these methods, the time-con-

suming, difficult method of ' clinical trial' of liver extracts would become more and more

obsolescent.

Experimental

Materials and samples.?We have analysed 110 samples of liver preparations for their vitamin B12 contents. The samples consisted

mostly of preparations for parenteral therapy, a few were oral preparations and they were both of foreign and Indian manufacture.

Test.?The basal medium is made of the

following ingredients :?

Acid hydrolysed casein to provide in the medium 0.16 per cent nitrogen.

Tryptophane solution (0.1 per cent) .. .. 100.0 ml.

Cystine solution (0.4 per cent) . . . . 25.0 ml.

Salt solution C .. .. 20.0 ml.

Dipotassium hydrogen phos- phate .. .. 2.5 gm.

Potassium dihydrogen phos- phate .. .. 2.5 gm.

Distilled water to .. 1,000.0 ml.

To this are added : uracil, adenine sulph., guanine-HCl, p-aminobenzoic acid, biotin, calcium-pantothenate, folic acid, nicotinic acid, pyridoxine-HCl, riboflavin and thiamine-HCl. Tween 80 is also added to the final medium.

The pH is adjusted to 6.9. The medium is dis- tributed in 9.0 ml. quantities in clean test-tubes of uniform size.

Salt solution C :

MgS04.7H20 .. 10.0 gm.

NaCl .. 0.5 gm.

FeS04.7H20 .. 0.5 gm.

MnS04.4H20 .. 2.0 gm. Water add .. .. 250.0 ml.

Shaw has added potato extract to the final

assay medium on advice from Cuthbertson, we have used instead tomato juice and find it an excellent substitute.

Glucose is added aseptically to the assay tubes. Tomato juice, glucose and the sample to be tested (or standard vitamin B12) are made

up in such a way as to be contained in 1.0 ml. The final volume of each assay tube is thus 10.0 ml. We had considerable difficulty in prepar- ing the casein hydrolysate as we could not get Labco vitamin-free casein. Most of the casein, due to the presence of traces of vitamins, supported the growth of L.L.D. even in the tube which contained no vitamin B12 and was used as the control tube. Subsequently we got good results with samples made from ordinary technical casein by first digestion with acid and subsequently autoclaving the sample under a

high pressure (30 lb. for 30 minutes). Culture for inoculation.?Lactobacillus lactis

Dorner is best maintained in soya-bean medium. Twenty-four hours before the test, subcultures are made on solid slopes of a medium contain- ing :

Peptone (Difco), purified liver powder, tween 80, glucose and tomato juice. The organisms are washed with sterile normal saline, centrifuged and the supernatant replaced with fresh normal saline. This is used for the inoculum.

Headings.?In arranging for a test, a set is

always included for the standard. As we could not procure a sample of standard crystalline B12r,

Sept., 1951] VITAMIN B12 AND LIVER EXTRACTS : DE & MUKERJI 405

we have been using Rubramin (Squibb) as our

provisional standard. After 24 hours' incuba- tion at 37?C., the sets are taken out and the

growth in the standard and the test-sample tubes are measured by turbidimetrie method.

Results.?Table I shows the number of

samples examined, and the results.

We have tried to screen the 110 samples under three different groups :

10 fig. or more/ml. or single Good dose *

5 fig. more or less/ml. or Fair

single dose Less than 2 fig./ml. or single Low dose

Table I

Number Preparation ^foreign?'

1 Injection I.

2 1 >1 F. 3 Oral (liquid) I. 4 ? (powder) F. 5 Injection I. 6 ? F. 7 ? F. 8 ? I. 9 ? I- 10 ? F. 11 ? I. 12 ? F. 13 ? I. 14 ? I. 15 ? I.

16 ? F. 17 ? I. 18 Oral (liquid) F. 19 ? (powder) F. 20 Injection I.

21 ? I-

22 ? I.

23 ? I-

24 ? I.

25 I ? I.

26 '

? F.

27 ? F-*

28 ? F* 29 ? F* 30 ?

F* 31 ? F. 32 ? I. 33 ? I. 34 ? F. 35 ? I. 36 ?

I- 37 ?

I. 38 ?

I. 39 ? F- 40 ?

F. 41 ? I- 42 ?

F. 43 ?

I- 44 ?

I. 45 ?

I. 46 ?

I. 47 ?

I- 48 ?

I- 49 ?

I. 50 ?

I- 51 ? I. 52 ?

I.

53 ? I.

54 ? I.

55 ? I.

Vitamin Bu

Preparation ^ MS-

Injection I. 2.5 I. 2.5 F. 1.0 I. 2.0 I. 2.0 I. 2.5 I. 2.5 I. 2.5 I. 2.5 F. 3.5 F* 0.5 F. 5.0 I. 5.0 I. 5.0 F. 7.5 F. 2.5 I. 2.5 I. 2.5 I. 2.5 I. 2.5 I. 1.5 I. 2.5 I. 2.5 I. 2.5 I. 2.5 I. 1.5

Oral (powder) I. 20.0

Injection F. 0.5

F. 2.5 F. 50.0 I. 2.5 I. 2.5 I. 2.5 I. 2.5 I. 1.0 I. 1.0 I. 1.0 I. 1.0 I. 2.5 I. 2.5 I. 2.5 I. 1.5

I. 2.5

I. 2.5

I. 1.5

F. 20.0

I. 2.5 I. 2.5

I. 2.5 I. 2.5 I. 2.5 I. 2.0 I. 2.0 I. 2.0 I. 2.0

?Date-expired samples.

406 THE INDIAN MEDICAL GAZETTE [Sept., 1951

Discussion

As will be evident from table I, the amount of vitamin B12 in different samples varied from 0.5 to 20.0 /xg./ml. The three samples showing more than 20.0 ng./ml. were preparations of crystalline vitamin B12 for parenteral therapy. From table II it will'be noted that out of 110 samples tested, 31 were of good quality, 57 were of fair (satisfactory) quality and 22 were con- sidered of low or definitely bad quality.

Table II gives the result according to this classification.

Table II

Foreign

Indian

Total

Good

20

11

31

Fair

4

53

57

Low

6

16

22

Total

30

80

110

N.B:?Where a single dose is 2 ml., the preparation is considered ' fair' if the total dose contains

more than or nearly 5 micrograms.

Cuthbertson ct al. (loc. cit.) and Shaw (loc. cit.) have found that usually a sample containing more than 10 fig. vitamin B12/ml.' or gm. gives good clinical response. In a

personal communication, Cuthbertson suggests that ' the doses recommended should not be less than 10 ng. at the beginning of the treatment and should not be less than 10 jug. B12 per week thereafter. Furthermore, one could suggest that liver extracts containing less than 1 to 2 /xg.

B12/ml. should not be recommended because of the large volumes of such extracts which would be required for adequate treatment'. In the United States, 1 jug. B12 is now considered as

equivalent to 1 original U.S.P. unit.

Accepting these suggestions we feel that a

fairly good screening arrangement would be

possible if we classify the preparations accord- ing to their B12/ml. or gm. basis. We suggest that all preparations containing-10 ng. or more

per ml. or gm. could be designated as '

good ', 5 ng. more or less per ml. or gm. or in single full dose of 2 ml. could be called a ' fair ' or ' satis-

factory '

preparation, whereas samples showing less than 2 /*g. per ml. or gm. should be rejected as

' bad ' samples. The classification in table II has been done

on this basis. It will be noted that out of 30

foreign samples, 20 were good, 4 were fair and 6 were of low or bad quality. Of these latter, 5 samples were date-expired items. Of the 80 Indian samples, only 11 were good, 53 fair and 16 were bad. The need for control of liver

extract preparations both imported and manu- factured in India becomes self-evident.

Judging from the fact that the micro-

biological assay (described in the paper) gives consistent results and also from the fact that

vitanjin B12 can be considered as one of the

principal anti-angemia factors, it should be

possible in future to screen the liver prepara- tions by this method. Under the Drugs Act, 1940, and the Drugs Rules thereunder, it could be made obligatory on all manufacturers and

importers of liver extract preparations to

declare the exact vitamin B12 potency on the label, carton, etc. This will at least help weed- ing out preparations which appear now to be

frankly ' below par

' in quality.

Conclusions

1. A microbiological method for estimating vitamin B12 is described for assaying liver extracts. This method has been found to be

sensitive, consistent and dependable. 2. The results of analysis of 110 different

commercial liver extracts are given and a

classification has been suggested on the basis of their vitamin B12 contents. Preparations con-

taining 10 jug./ml. or more are considered '

good ', 5 jug./ml. more or less as ' fair ' and

those below 2 ng./ml. are considered 'bad' and not acceptable for clinical use.

Our sincere thanks are due to Dr. Cuthbertson of the Glaxo Laboratories for his valuable suggestions and advice and for the strains of micro-organisms and samples of liver extracts which he supplied. The

Director, Haffkine Institute, Bombay, and Dr. B. P. Ghosh of Gluconate Ltd., Calcutta, helped us by giving us certain essential ingredients for the media.

REFERENCES

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Cuthbertson, W. F. J., Biochem. J., 44, iv. and Smith, L. (1949).

Editorial (1949) .. Lancet,.ii, 565.

Ghosh, D. P., and Indian J. Med. Res., 38, 269. Mukerji, B. (1950).

Hall, B. E., and Camp- Proc. Staff Meet. Mayo bell, D. C. (1948). Clin., 23, 591.

Muller, G. L. (1935). New England J. Med., 231, 1221.

Rickes, E. L., et al. Science,' 107, 396. (1948).

Shaw, G. E. (1949) .. J. Pharmacy and Pharm., "f? 695 and 701.

Shorb, M. S. (1948) .. Science, 107, 397.

Smith, E. L. (1948) .. Nature, 161, 638.

Spies, T. D., et al. South. Med. J., 41, 522, 523. (1948). Idem (1949) J. Amer. Med. Assoc., 130,

521.

West, R. (1948) .. Science, 107, 398.