INDUSTRIAL
AND
ENGINEERING CHEMISTRY PUBLISHED B Y THE A M E R I C A N C H E M I C A L SOCIETY WALTER J. M U R P H Y , EDITOR
Physiological Availability of the Vitamins Study of M e t h o d s for Determining Availability of Vitamins in Pharmaceutical Products BERNARD L. OSER, DANIEL MELNICK,
AND
MELVIN HOCHBERG N. Y .
Food Research Laboratories, Inc., Long Island City, The nature of the precautions taken to ensure the stability of the vitamins in pharmaceutical capsules or tablets, which are usually swallowed whole, raises the question of the physiological availability of the vitamins. The limitations of in vitro and in vivo disintegration tests and of serial roentgenograms after oral administration are discussed and the possibility of drawing erroneous conclusions from such studies is pointed out. The new bioassay technique for the direct determination of the availability of vitamins to man is dependent upon correlating the urinary excretion of the water-soluble vitamins (or derivatives) with vitamin intake. Experimental support i s advanced For the many advantages ot the new procedure.
dosage. Here the assumption has been that shadows resembling tablets or small opaque areas which might be fragments of tablets actually represent undisintegrated and hence unavailable portions of the vitamin content. It has recently been reported from the authors’ laboratories ( 7 ) that the urinary excretion by man of the water-soluble vitamins resulting from standardized conditions of dosage may be employed as a means for estimating the availability of these factors. The urinary excretion of these vitamins as such (or as their derivatives) parallels the quantity consumed, provided that normal subjects are employed and that a t the time of the tests they consume an adequate diet. For the purpose of calculating the degree of vitamin availability, comparison is drawn between the extraurinary excretion after taking the test dose and that following the oral administration of an aqueous solution of the pure vitaminLe., the form in which the vitamin is regarded as most readily available for absorption. The assay technique, its reliability, and applicability to studies in human nutrition have been discussed in detail ( 7 ) . This report is concerned with investigations on the reliability of the techniques mentioned above for determining vitamin availability in pharmaceutical products, fhree being selected for illustrative purposes. Multivitamin-mineral Tablet A contained vitamins 4 and D and the water-soluble factors riboflavin, nicotinamide, thiamine, and ascorbic acid, only the last two being coated with a water-insoluble enzyme-resistant material. In Tablet B all these vitamins were present but all the water-soluble vitamins were protectively coated as in Tablet A. In the BComplex Capsule E the thiamine and riboflavin were present in the form of a fuller’s earth adsorbate. In this report is also described an indirect procedure for estimating the availability of the fat-soluble vitamins in multivitamin products.
B
EC.1USE of the numerous and obvious advantages of chemical and microbiological mcthods for the determination of vitamins in foods and pharmaceutical products, the need for biological assays is becoming less imperative. Nevertheless the biological assay has a unique advantage-namely, it measures the available or potentially effective portion of the vitamin content, whereas the nonbiological methods measure the total quantity of vitamin present. The War Food Administration, probably the largest single purchaser of vitamin products in the world, has recently specified that the vitamins in products purchased by it not only be present in sufficient total quantities (1) but be in a form completely available to the human when the product is swallowed whole. In view of the fact that there was no assay technique for measuring availability, a simple in vitro digestion test was specified. This procedure can lead to false conclusions, as is shown below. (Obviously rat assays cannot be employed for this purpose. Larger laboratory animals, which are capable of swallowing intact tablets or capsules, likewise cannot be used, since the vitamin intake would far exceed the critical range of dosage in the conventional prophylactic or curative bioassays.) A recent seizure action by the Food and Drug Administration was based upon the recovery from the stools of human subjects of intact vitamin tablets, from which it was justifiably assumed that the vitamins were not available. However, the converse observation-namely, disintegration of the tablets in the gastrointestinal tract-would not necessarily prove the vitamins available. Most tablets, particularly of the multivitamin-mineral variety, contain a sufficient quantity of mineral salts to make them opaque to x-rays. Attempts have been made to determine the availability of vitamins in such tablets swallowed whole by taking serial roentgenograms during the 24-hour period following
EXPERIMENTAL
Five nutritionally normal males were employed in the urinary excretion studies. Deficient subjects cannot be used because they retain extradietary vitamin and hence it is not possible to obtain a reliable ratio of extraurinary excretion as an index of availability. The subjects selected for test subsisted regularly on adequate well-balanced dietaries and excreted the water-soluble vitamins (or derivatives) well within the normal range both before and after postprandial dosage with extra vitamins ( 7 ) . The paper just cited describes the method employed for the quantitative collection of urine samples, the selection of analytical procedures, and the composition’of the basal diet eaten a t the time of test. 405
46
INDUSTRIAL A N D ENGINEERING CHEMISTRY
VoI. 17, No. 7 STUDIES WITH MULTIVITAMIN. MINERAL TABLET A
Tablet A was employed as the test materisl for the initial and more exhaustive part of this study of vitamin avdability. Fifteen of these
vitml’s in aqueois solution ,r>H 31 were also taken posk pruuJially. T h e urine .imi,lr.s collected durinc r w h 2 4 h k Derind subsequ&t to dosage keiz partitioned in order t o yield data. ou the rate of ahmmtion and excretion of the’ water-soluble vitamins. By subtracting the 24-hour excretion value8 when the subjects subsisted on the basal diet alone from the values obtained for the rorrcaponding periods follon,ing vitamin dosage, the in-
~ r s m e n t due s 4 e l v I Ornkine
the- extradietsry. vitam& were obtained. The urine samples were evlleetell lor the h i , wcond, m i l tliird4hour orriod.i a i d ior t1.e tinill 12-hour period after dosage. Roentgenograms were taken before and immediately after swallowing the tablets and 4. 8. 12. and 24 hours tbere-
Figure 1. Serial Roentgenograms of Subject D.M.
I n Table I the test doses of extra vitamins are shown. According to the chemical analyses, the vitamin content of fifteen tablets approximately equaled that of the pure solution. [ T h i a m i n e was determined by the thioc h r o m e p r o c e d u r e (3). ascorbic acid titrimetrically (6),riboflavin by the fluorometric method (S), and nicotinamide colorimetrically (8).] The di5erences are of nosignificance, since the comparisons are based on percentage excretions of the test doses and i t has been demonstrated (7) that a t the dose levels employed the relationship between dosage and urinary excretion is linear. I n making up the “solution dose” the f&soluble vitamins were kept separate from the B-complex solution and these in turn from the ascorbic acid solution. All solutions were prepared just prior to their ingestion immediately after the main or midday meal. A two weeks’ period intervened between oontrol and test periods. The tablets were swallowed whole, allowing
ANALYTICAL EDITION
July, 1945 Table
I.
Preparation of Pure Solutions of Vitamins
I = gelatin capsule
MQ. Thiamine Ascorbic acid
\
(28,000 U.S.P. units of A 2,800 U.S.P. units of D
50 mg. of thiamine
0.394 11.5
Water (pH 3) 1000 cc.
Test dose, 15 intact tablets
from the tablet was somewhat slower than from the pure solution. I n the case of the thiamine this may actually be desirable, because the total quantity of extra thiamine absorbed and consequently excreted following tablet dosage was consistently greater than that following the administration of the pure solution. I t appears probable that under these circumstances less thiamine was destroyed i i the intestinal tract (6, 9) prior to absorption The data in Table I11 also indicate that whereas riboflavin taken in pure solution was rapidly absorbed and excreted, there was a significant lag when the vitamin was taken in the form of the intact tablet, the values for the first 4-hour period being relatively small. However, during subsequent periods the rate of riboflavin absorption increased markedly, so that 24 hours after dosage the same percentage of the test dose was found in the urine as when the pure solution was taken (see Table IV). The results obtained in the riboflavin experiments are consistent with the low solubility of pure riboflavin. In Table IV are summarized the results of the serial roentgenograms of the test subjects taken periodically after the tablets had been swallowed whole. For illustrative purposes typical roentgenograms obtained with subject D.M. are presented (Figure 1). I n Table IV the word “tablets” is written in quotation marks fqr the reason that the circular shadows on the x-ray have been uncritically assumed by some roentgenologists to be intact tablets. The following observations prove the error of this interpretati on :
Test Dose of Extra Vitamins
Vitamin Content of Tablet U.S.P. unit8 Vitamin A 1960 Vitamin D 196
to
Test dose, one capsule of I; 100 cc. of 11; 20 cc. of 111
Vibmlnr Yielded by Test Dose
Tablets U.S.P. units 28,400 2,840
Vitamin A Vitamin D
Mg. 5.91 173.0 10.6 55.0
Pure Solution U.S.P. units 28,000 2,800 MQ.
Thiamine Ascorbic acid Riboflavin Nicotinamide
401
5.0 200.0 10.0
50.0
Seven of the 15 tablets (average for the five subjects) were the normal digestive processes to disintegrate them and liberate clearly visible in the 4-hour roentgenograms. This is a minimal figure, since others may have been present but concealed by their contents for absorption. tissue. However, the urinary excretion of the vitamins or OF cRINa& E~~~~~~~~kUDIEs. ~ ~II presents b l ~ bone derivatives during the second 4-hour period was considerable, the results of experiments on the availability of the water-soluble approximating that noted during the first 4-hour period followvitamins derived from the intact tablets. They demonstrate that ina the taking of the Dure solutions. This indicated that 4 hours these vitamins are as completely available when taken in the intact tablet as when administered in pure solutions. Table 11. Availability of Water-Soluble Vitamins in a Multivitamin-Mineral Tablet The data indicate the thiamine availability to be 122%. This apparently Control Period Test Period
-
greater-than-theoretical availability of thiamine is not an artifact; it has been consistently observed in other assays of this and similar tablets from the same source. In the case of nicotinamide, measurements are made of the urinary N1-methylnicotinamide, the main excretory product of nicotinamide dosage.
Vitamin Ascorbic acid
J.C.
E.M.
D.M.
M.H.
H.H. Av.
CORRELATION OF URINARY EXCRETIOX VALUES WITH SERIAL ROENTGEEOGRAMS.Thiamine The 24hour urine samples collected during the control and test periods, following dosage with pure solution and tablets, respectively, were partitioned in order to determine the relative rates of vitamin availability. These data were of value in interpreting the x-ray findings. The average urinary excretion results obtained for the four water-soluble vitamins are presented in Table 111. The values following dosage were corrected for the basal figures for the corresponding test periods and the results for each period listed as per cent of the total 24-hour excretion. In the case of ascorbic acid, thiamine, and nicotinamide, the major urinary excretion of the test doses occurred during the first 8 hours and was followed by a progressive decrease. The rate of absorption of these vitamins
Subject
J.C.
E.M.
D.M. M.H. H.H. Av.
Riboflavin
J.C. E.M. D.M. h1.H. H.H. .4v.
Nicotinamide
Excretion, Mg. per 24 Hours Basal After dosage
Excretion, l l g , per 24 Hours Basal After dosage
% dose excreted
Dose, 200 hlg. of Ascorbic 183 41 57 159 47 131 70 162 28 93 49 146
Acid Dose, 15 Tablets (173 Mg. of Ascorbic Acid) 63 35 116 47 59 18 92 43 42 38 128 52 46 96 46 l7 109 52 33 20 49 26 108 48
.
Dose, 15 Tablets (5.91 Mg. of Thiamine) 0.33 2.01 28 0.42 1.61 20 0.39 1.62 21 0.27 1.52 21 0.39 1.69 22 0.36 1.69 22
Dose, 5.0 Mg,of Thiamine 0.16 0.87 14 0.31 1.23 18 0.25 1.10 17 0.21 1.22 20 0.19 1.13 19 0.22 1.11 18
Dose, 15 Tablets (10.6 Mg. of Riboflavin) 0 68 5.12 42 0.95 8.17 68 0.69 5.79 48 0.84 7.20 60 0.74 6.83 57 0.78 6.62 55
D&e. 10.0 ?dg, of Riboflavin 0.69 4.95 43 0.54 7.05 65 0.59 5.43 48 0.72 5.37 46 0.55 6.71 62 0. 62 5.90 53
Dose, 15 Tablets (55.0 Jlg. Nicotinamide) 6.2 17.9 5.9 18.8 6.7 16.6 4,s 12.5 3.0 14.7 5.2 16.1
Dose, 50.0 \Ig. of Nicotinamide J.C. 5.8 20.5 29 E.M. 6.2 20.5 29 D.X. 4.1 13.9 20 M.H. 5.2 10.4 10 H.H. 3,s 11.2 15 Av. 5 , 3 15.5 21
-
Availability Ascorbicacid 48/49 X Thiamine = 22/18 X Riboflavin 55/53 X Sicotinamide 20/21 X F
52 dose excreted
100 100 100 100
= 98Yc = 122% = 104Tc
=
95%
of 21 24 18 15 21 20
408 Table
111.
Rate
of Urinary Excretion of Water-Soluble Vitamins
(Following oral dose of pure ~ q u e o u asoldtins and intact tablets expressed as per eent of 24-hour excretion figures‘) Periodic Excretion5 Last First Seoond Third Dosage 4. 4 4 12 hour? hours hours Vithmin From Quantity hours MQ. Me. Mo. Mg. Mg. 25 30 11 34 200 Pure solution Ascorbic acid 19 36 20 26 173 15 tableta
{
Thiamins
i
RiboEavin
Nicotinamide’
Pure solution
5.0
45
35
6
14
15 thblets
5.9
17
45
23
14
Pure solution
10.0
56
21
8
15
15 Tablets
10.6
9
35
21
35
Pure solution
50.0
40
14
12
33
15 tablets
55.0
28
30
8
40
I
o. Correction made for b a d excretion values when sthndardiaed diet alone WBS inze-ested. a Average values for five tpt 8Fhiecb. e Excreted aa N’-methylnlcotmsmlde.
Table
IV.
Serial Roentgenogrsrnr of Subjects Following Tablet
-
DOSalle
sub.
Seen on X-Ray Films after D O S B ~ 4 Hours 8 Houra 12 hours
awpab. lets” J.C. 9 E.M. 6 D.M. 6 W.H. 9 H.H. 5
ject
Vol. 17, No. 1
INDUSTRIAL A N D ENGINEERING CHEMISTRY
~ ~ ~“Tabg mentS lets” 0 1 3? 0
.Fragmenta
“Tablets”
?:
0 0
0
2
0
1
2 1
0
‘ 1
0
1
0
4
Fragmenta 5? 0 0
0 0
24 home “Tab- Fr%lets” menta
0
0
0 0 0
0 0 0
0
0
after tablet dosage the vitamins are in solution to the same extent as those in t h e , cqntrol solutions taken postprandially. Remembering that t h u IS over and above the quanti1 ties excreted during the first 4 hours after the intact tablets were ingested, i t is hardly possible that such largequantities of vitamins could have been excreted while one half ai the tablets were still intitct. The only likely explanation of this seemingly paradoxicalsituation is that the shadows seen on the x-ray films are not those of intact tablets but rather tablet residua
lets. The disintegration test was that described by the War Food Administration (1) in specifications for vitamin tablets (or capsules). Multivitamin tablets, pills, and capsules, sf,,. pended for 2 hours on a 16mesh wire screen in artificial gastric fluid a t 37’ C. and then similarly suspended for 2 hours in rtrtificia1 intestinal fiuid a t 37” C., shall disintegrate sufficiently SO that the active ingredients will be completely dispersed. The artificial intestinal fluid may be slight1 agitated to aid dispersion a t the end of the second %hour perioz. lyl-
The artificial digestive fluids for these tests were prepared according to prescribed formulas ( 1 ) . From Figure 2, showing the state of disintegration of the tablet, it is apparent that a t the end of the %hour suspension of the product in the artificial gastric juice only superficial disintegration had occurred. However, within 0.5 hour after the gastric juice had been replaced by the alkaline intestinal fluid, marked disintegrstion of the tablet occurred, much of the inner granular material having being washed out. After a full hour of “intestinal” digestion, hollow, opaque shells remained, the material adherent to each shell being of a loose granular consistency, so that with agitation i t could be removed. At the end of the second hour of “intestinal” digestion the tablet residua or “shells” were extremely thin and fragile and could be broken into small fragments merely by gentle tapping. I n Figure 3 is presented a series of roentgenograms showing the opacity of the tablet during the oourse of in vitro digestion. This readily accounts for the appearance of shadows on the roentgenograms ofthe test subjects which suggested the presence of undigested tablets, despite the fact that the vitamins (or derivatives) were being excreted at a rate only slightly less than that noted with pure solutions. Obviously, therefore, it is errone-
P
3
5
6
Support far this conclusian was found in the in vitro disintegration tests supplemented by periodic photographs and roentgenog r m s of the digested tab-
_?)
4 Figure
P.
Tablet
A Subjected to in V i t r o Dirintogrstion Test ( 1 )
ANALYTICAL EDITION
July, 1945
409
nrid, and nicotinamide are SO soluble t l a t retardation of the rsk? of solution i oithesevitarninsbecau~eoftheorganic iirotertive costing docs not interfere with their availability. The possibility existed tbrtt an appreciable part of the extmuinary excretion of riboflavin would occur during the second 2 4 h m r period following dosage with these tablets. Tests on the subsequent urine samples showed 1 P 3 4 that the e&a riboflavin found in the Figure 3. Opacityto X-Rays of Multivitamin-Mineral Tablet during in Vitm Digestion %hour wecimens. and above the basal excretions, was 9% of Tablet viewed through superimposed hand 1. Original tablet the control dose of pure riboflavin soluP. Garhic Idee P hova 3. Gashic juice P hovs, pancreelic iuice 1 hour tion and 5% of the tablet dosage. 4. Gastric juice P hours, pancreatic inice P hours Thus, calculation of the riboflavin avrtilability based upon the total 48-hour excretion gave the same value as was found for the first ous for the roentgenologist to conclude that such shadows repre24 hours. sent undisintegrated tablets and hence unavailable vitamins. Chemical analyses conducted an the tablet shells have shown I n many studies conducted in these laboratories both on the them to be almost completely free from ascorbic acid, thiamine, availability of vit,amins in various products and on conditioning riboflavin, and nicotinamide, containing less than 1%of the vitafactors (4) which may reduce the availability, analyses were frequently conducted on the urine samples collected during the mins in the original tablet. second as well as the first 24hour period following dosage. I n Since these tablets did not disintegrate completely in a static no case did the carry-over values (second 24-hour excretions) in vitro digestive system ( I ) , doubt is cast upon the validity of alter the interpretation of the data based solely upon the figures wch a disintegration test as an index of availability. Significant obtained for the 6rst 24-hour samples. If vitamin absorption from the gastrointestinal tract is to be adequate, i t must occur also in this connection are the loose consistency of the contents of well within the first 24 hours following dosage. Otherwise, the the shells and the apparent absence of tablet particles from the vitamins are excreted in the stools and lost to the organism. stools. The assays of this product were repeated and confirmatory reSTUDIES WITH MULTIVITAMIN-MINERAL TABLET B sults obtained; the availability of the riboflavin was now found to be 51%, whereas again no interference in availability of the Urinary excretion studies were conducted on the avdability other water-soluble vitamins was noted (see Tsble VII). When of the water-saluble vitamins in Tablet B in which all vitamins these tablets were chewed, rather than swallowed whole, there were protectively coated witb a water-imoluble enzyme-resistant was an increase in the quantity of riboflavin absorbed from the film. It was found that coating the more soluble components, gastrointestinal tract, the availability being 75%. Reducing ascorbic acid, thiamine, and nicotinamide, did not interfere with the tablet dose to one third, but still swallowing the product their availability. However, in the case of riboflavin, there was whole, gave a value of 85% riboflavin availability. The limited a marked decrease in the urinary excretion values following solubility of the vitamin in the digestive juices, as well as the tablet dosage, indicating that only a fraction of the riboflavin additional possibility that the protective coating interfered with was absorbed from the disintegrated tablet during its passage the extractability of this vitamin, may have been responsible through the gastmintestid tract. The riboflavin excretion for its lower availability when the 15-tablet dosage was adminisdata presented in Table Vindicate that 55% of the riboflavin in tered. Another batch of tablets was prepared in the ssme manthis tablet was available for absorption. The responses among ner except that this time the riboflavin was not coated. Complete the various subjects during control and test periods were strikavailabili4y (15-tablet dosage) was now attained, the figure of ingly uniform. 91% being within the experimental error of the assay technique. The most obvious explanation of the lower availability of the Roentgenograms of these tablets in the eoume of digestion in riboflavin is its limited solubility in water. Thiamine, ascorbic vitro and in vivo were similar to those for Tablet A. In no case were tablets or tablet residua recovered from the stools. Therefore disintegration in vivo cannot be regarded done as indicative of complete availability. When these tablets were subjected to Table V. Availability ol Coated" Riboflavin in Multivitaminthe War Food Administration test ( 1 ) the same results were obMineral Tablet tained witb Tablet B as with Tablet A. Despite the fact that Control Period, Pure Solution Test Period. 15 Tablets (10.0 Mg. of Riboflavin) (11.8Mg. 01 Riboflavin) unequivocal evidence of interference in riboflavin availability was Basal After Test Basal After Test obtained in the urinary excretion studies, this in vitro test failed exmetest dose exeretest doae Bubjeot tion dose excreted tion dose excreted to differentiate between these two types of tablets. Thns, the difference in the availability of the ribotlavin in Tablets A and B J.C. 0.77 is not reflected either by roentgenographic studies oraby simplp 6.04 53 0.77 5.63 41 E.M. 0.82 7.12 63 0.76 4.08 28 disintegration teste whether conducted in vivo or in vitro. D.M. 0.76 6.24 55 0.54 3.47 25 ~
M.A.
H.H. Av.
0.81
0.87 0.81
6.07 6.29
53 54
6.35
56
Availability of riboflavin in tsblets
0.69
3.99
1.03 0.76
4.70 4.37
-$
X 100
-
28 31
31 55%
Watcrtloluble vitamins coated witb water-inaoiubie enzyme-resistant mnteria1.
STUDIES WTH ECOMREX CAPSULE E
Adsorhents rue frequently used in pbarmaceutical preparations for one reason or another. The effectivenessof certain adsorbents in removing B vitamins was widely utilized for the concentnrtion and isolation of these factors from natural sources. An acid-clay adsorbate of vitamin Bz was formerly emdoved as the reference
410
Vol. 17, No. 7
INDUSTRIAL AND ENGINEERING CHEMISTRY
inaterial for bioassay purposes, thc international unit being dcfined as the antineuritic activity of 10 mg. of a standard adsorha t e (IS). I-Ion.ever, subsequent studies ( f f , IS) h a w shown that this matorial, ai1 international standard, contained actually twice as much thiamine as was helieved to bc present bccauso duriirg digestion by the rat only half of the thiaminc was clutcd.
VI. Availability of Thiamine in B-Complex Capsule
Table
Containing Fuller’s Earth
hbject
Control Period. Pure Solutio11 ( 5 . 0 M g . of Thiamine) Basal After Test excretest dose tion dose excreted iMg.,’-‘4 hoicrs
J.C. E.N. D.N.
0.20 0.23 0.30
1I.H.
H.H.
Av.
0.2G 0.24 0.25
c
Test Period. 3 Capsulei” L . 0 Afg, of Thiamine) Basal After Teht excretest dose tion dose excreted .111J./;24 h0ur.s (/ U’
1.08 1.00 1.35 1.50 1.39
21 25 23
0.24 0.27 0 29 0,21 0.23
1.26
20
0.25
18 1 .j
0.41
1.06 0.56 0 59 0.48 0.63
3 1G 6 8 5 8
-
Availability of tlliniuine in capsules = 8 X 100 = 405;
20 Each containing 333 mg. oi fuller’s earth as a riboflavin adsorljent.
Considerable difficulties were encountered a t one tiinc in chemical analysis of a B-complex capsule containing a fullcr’s earth adsorbate. Exhaustive strong acid extraction was neccssary to obtain results in agreement with the claimed quantity of thiamine. This led to the suspicion that. the vit,amin in this capsule might not be completely available to the human. The results of the urinary excrctipn tests in which five capsules wcrc taken postprandially by each of the test subjects are presented in Table VI. These indicate that the availability of thiamine in this capsule was very low, the average for the five subjects being 40%. It is of interest to note that the findings for one of the subjects indicated his ability to effect complete elution of the thiamine from the fuller’s earth adsorbate. The study was rcpcatcd with this individual and essentially the same results were obtained. This variability in the responses of the human subjects finds its counterpart in the variability in thc responses of rats in the old biological assay procedure when the standard adsorbate was employed as the reference material. In order to ascertain whether the apparent low availability of the thiaminc was due to a slow rate of absorption of the vitamin as evidenced by a delay in its excretion, analyses were conducted on the urine samples collected during the second 24-hour period following dosage. The “carry-over’’ values showed the same relationship to each other as those of the first 24-hour samples. The low thiamine availability must thcreforc be attributed to incomplete elution. Similar studies conducted with the adsorbate indicated that only 79% of the riboflavin present was available. In vitro tests furnished additional evidence in support of the results obtained in the human bioassays. Thiamine was found to be more readily adsorbed and more firmly retained by the adsorbent than riboflavin. When the vitamin capsule was subjected to the disintegration test of War Food Administration ( I ) , the gelatin shells were readily digested, leaving the contents on the screen as a soft coherent map. With agitation the particles were readily dispersed, S O that the capsule would have passed the disintcgration tcs;t. The capsules were opaque to x-rays, although during digestion their contents soon became diffused and indistinguishable. That) this indication of disintegration could not be accepted as proof of availability was demonstrated by the finding of only partial availability of the B vitamins in the urinary excretion tests. The results of the studies with this capsule illustrate again thc limitations of serial roentgenograms and of simple in vitro or in vivo digestion tests as indexes of vitamin availability.
DISCUSSION
I n Table \’I1 is presented a summary of the availability studie3 conducted on a number of pharmaceutical products. An index of the reproducibility of the technique is obtained from an examination of the results obtained in repeated assays of the same product. These data serve to emphasize that in the case of Tablet A enhanced thiamine stability in vivo occurs, so that a greater fraction of the test dose is absorbed (and excreted) following tablet dosage. That thiamine may evidence varying degrees of stability in the digestive tract has been demonstrated (6, 9). The results obtained in test Tablet B, particularly with reference to riboflavin availability, are in consistent agreement with the low solubility of this vitamin in aqueous solutions. The values obtained n-ith Capsule E, containing the fuller’s earth adsorbate, find experimental support in rat assays of similar adsorbates. The results of the present study demonstrate that of the various assay techniques employed, the only reliable procedure for estimating vitamin availability is the one based upon urinary excretion measurements. By this technique the availability of specific vitamins may be determined and assays for a number of factors conducted all at the same time. It has been found in the course of these studies that the control responses by the standardized subjects following pure solution dosage are reproducible (within *lo%), so that the control tests need not be repeated each time ( 7 ) . Thus, for subsequent routine assays conducted on Tablet -4an experimental period of only 2 days was required, the remainder of the week being devoted to analyses of the urine. It has also been possible by collecting the urine samples a t the end of consecutive 4-hour periods to evaluate rate as well ail degree of vitamin availability.
Table VII.
Summary of Availability Studies Conducted on Pharmaceutical Products
.4vailability Ascorbic Thisacid mine Per 98 122 .\Iultivitamin-~IineralTahlet tia Same b u t from another batch 102 182 98 117 Same h u t from another batch Same but from another hatch 96 120 Sample
.\lultivitarnin-~llineral Tahlet B, riboflavin coatedb Same but from another hatch Same b u t tahlets chewedC Same h u t tablet dose reduced t o 33% Same but riboflavin in tahlet uncoated ZIultivitamiti-.\Iineral Tablet C, riboflavin coatedd
\Iultivitamin Tablet D* 13-Complex Capsule E , containin: fuller’s ea1 th adsorbate
115
86
...
... ...
... 96
of Vitamins RiboNicotinflavin amide
cent 104
106
102
10.5 81 80
..
..
55 51
100
... ...
85
...
91
...
106 113
...
,
75
107
...
..
107
...
141
98
11.5
35
79
,
Comniercial tablets of same manufacture. b bVater-insoluhle enzyme-resistant organic material employed in coating iill water-soluble vitamins. C For this assay alone tablets chewed before swallowing; in all others tahlets taken whole. d Rihoflavin coated, with water-insoluble organic material readil!. soluliilized h!, digestive fluids. a
Various simplifications in technique suggest themselves. I t should be possible to pool the urine specimens from the five subjects and conduct the analyses on just the composite sample. Since calculations of the availability of vitamins in pharmaceutical products are based entirely upon the extra excretion following test dosage, simplifications of the methods employed for the analyses of the urine samples are justified, For example, the titrimetric procedure for ascorbic acid ( 5 ) may be employed, sincc the calculations of the extraurinary excretion values automatically correct for the presence of nonspecific reducing substances in the urine samples obtained during the two consecutive periods
ANALYTICAL EDITION
July, 1945
Analyses may be run for only one factor, preferably riboflavin, the least soluble of the factors, to yield data indicating tablet disintegration in vivo. However, the results of such tests must be interpreted with caution if deductions are to be drawn on the availability of the other factors. Because fat-soluble vitamins are not normally excreted i n the urine, the procedure here described is unsuited to the determination of their physiological availability. An indirect, but nonethcless sound, approach to this question can be made by correlating bioassay experience with analytical data (Table VIII). In the manufacture of sugar-coated tablets, the central core uaually receives a moisture-repellent coating of shellac. This shellac film is indigestible and may contain as much as 10 to 15y0 of the vitamin A as determined by colorimetric analyses (10) of the shell residua remaining after an in vitro disintegration ( I ) . Severtheless, bioassays (12) of ground whole tablets and, what is more critical of the ground tablet residua, fail to show less than complete availability to the rat of such vitamin A or Il as may be contained in the shellac coating. In order to feed assay closw of vitamin tablets to rats it is necessary first to grind the tablcts. A finding that whole tablets actually disintegrate to the same degree in human digestive fluids would constitute indisputable evidence that this preliminary disintegration for assay purposes is a justifiable practice. However, it could be argued that a finer state of subdivision is achieved by the prtliminary grinding of tablets or shell residua for assay than might normally occur i n the human intestinal tract. That this argument is untenable is shown by the fact that the efficiency of in vitro extraction of vitamin A from tablet residua by repeated washings with the digestive fluids was not influenced by the degree of subdivision. In other words, even lvhen finely ground, these shellac films or shells retain a residuum of vitamin A whirh is resistant to further in vitro extraction. Thus, in tablets whose water-soluble vitamins are shoum by urinary excretion studies to be completely available, the availability of the fat-soluble vitamins is supported by the facts that (a) the tablets disintegrate in the human digestive tract, ( b ) bioassays for vitamins A and D indicate that the rat is capable of extracting them completely from ground tablet residua, and ( c ) the vitamin A remaining in the shells after repeated in vitro extraction is independent of the state of subdivision. It is of course inconsistent with these observations to accept as physiologically available all the vitamins in a tablet merely because its fragments after an in vitro digestion test are small enough to pass through a screen of arbitrary mesh; on the other hand, failure to disintegrate into small particles does not constitute ipso facto evidence of nonavailability. SUMMARY
The need for methods for the determination of the availability to man of vitamins in pharmaceutical capsules and tablets, particularly when these products are taken intact, has recently been emphasized. Simple in vitro and in vivo disintegration tests and roentgenograms taken serially after dosage with the test product have proved to be of only limited value and may even lead to misinterpretation. The only suitable procedure for estimating the availability to man of the water-soluble vitamins in pharmaceutical products is that based upon measurement of the urinary excretion of the vitamins (or derivatives) following dosage. The urinary values obtained with normal test subjects parallel the vitamin intake. For the purpose of calculating the degree of vitamin availability, comparison is drawn between the extra urinary excretion following dosage with the test material and that following administration of the vitamins in pure aqueous solution, the form in which they are considered to be most readily available for absorption. It has been demonstrated that vitamin availability may be reduced even in t,ablets or capsules which disintegrate in vitro or
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i n vivo, and that one vitamin may be completely available for absorption while another is only partly available. By the new bioassay technique, described in this report, it is possible to carry out simultaneous tests for four vitamins (ascorbic acid, thiamine, riboflavin, and nicotinamide); to evaluate rate as well as degree of vitamin availability; to conduct the tests directly on human subjects; and, when the responses of the test subjects have been sufficiently standardized to pure solution dosage, to complete the availability study on a given product within as short a period as one week. The precision of the assays is superior to that of most animal tests. An indirect procedure is described for estimating the availability of the fat-soluble vitamins in multivitamin products. Various simplifications in the assay techniques are suggested.
Table VIII. Availability of Fst-Soluble Vitamins" in MultivitsminTablet D Experiinen t
Results
Conclusions
.4vuilability t o man of water-soluble vitainins in intact tahletv
Ascorbic acid, 9 6 % ; thiamine, 141 %; riboflavin, 9 8 % : nicotinamide 115%
Tahjets disintegrate in
R:$t biological assays for vitamins A and D on ground tablets
I'itamin A , not less than 3500 units per tablet; vitamin D , not less than 200
I'reauniptive evidence for availability of fat-soluble vitamins
\'itamins i i i table residua after in vitro rlisintegratiori test ( I )
\Vater-soluble vitamilis. less than 1% of total. Vitamin Ab = 308; vitamin DC = 30 units per tablet residuum
Shellac coatings in t a b let? retain 1270 of vitamin A and 15% of vitaininD
vitamin A b in tablet residua after repeated washings of particles, varying in sized
Uiibroken half shells = 300 units per tablet residuum: washings = 4. 5-mesh particles = 360; r a s h ings = 3. Fine par!irlese = 292; washlngs * I
Degree of subdivision of particles is no index of atnountsof fat-soluble vitamins retained. Interpretation of availability based p n simple W.F..\. disintegration experimental test (I) lacks
I h t biological a s h a ~ - s Yitainin A = = 300; for vitariiins A and D vitamin DC = 30 on ground tahlet reunits per tablet residuum sidua
Fat-soluhle vitaiiiiiis in shellac coatings nre nvailnhle
vivo
support
Specificatioi~scalled for 2900 U.S.P. units of vitamin .\ and 300 C.S.E' uiiits of vitamin D per tablet. b Test conducted on uiisaponifiable extract by colorimetric procedure ( 1 0 ) . C Multiple level rat assay (12). d Tests conducted on three samples of tablets. After enzymic digestion Gesjdua were ground (where indicated) and washed with digestive fluid-. ,uitable for feedingin rat assay ( 1 2 ) .
LITERATURE CITED
(1) Federal Surplus Commodities Corp.,Form SCP-1738, ProgramGCP (1944). Hennessy, D. J., IND.ENG.CHEM., ANAL.ED.,13, 216 (1941). Hodson, A. Z . , and Norris, L. C., J . Biol. Chem., 131,621 (1939). JollifTe, N., J . Am. .Wed. Assoc., 122, 299 (1943). King, C. G., IKD.ENG.CHEM., ANAL.ED.,13, 225 (1941). (6) Melnick, D., Field, H., Jr., and Robinson, IT. D., J . .Yutrition, 18, 593 (1939). (7) Melnick, D . , Hochbeig, M,,and Oser, B. L., Ibid., in press. (8) Melnick, D., and Oser, B. L., IND. ENG.CHEM.,.%SAL. ED., 15, 355 (1943). (9) Melnick, D., Robinson, W. D., and Field, H., J r . , J . Biol. Cheni., 138, 49 (1941). (10) Oser, B. L., Melnick, D . , and Pader, h l . , ISD. ESG. CHEM., h N h L . ED., 15, 724 (1943). (11) Sampson, J. C.,and Keresztesy, W.L., Proc. Soc. Ezpll. Biol. .!fed., 36, 686 (1937). (12) C. S. Pharmacopoeia XI1 (1940). (13) Williams, R. R., and Spies, T. D., "Vitainin BI (Thiaiiiine) and Its Use in Medicirie", New York, Macmillan Co., 1939. (2) (3) (4) (5)
PRESESTED in p a r t before the Division of Biological Chemistry a t t h e 108th Meeting of t h e AMERICAN CEIEMICAL SOCIETY, New York, N. Y. One in a series of papers dealing with t h e physiological availability of t h e watersoluble vitamins. T h e expenses of these studies were defrayed by a grant from Lever Brothers Co., Cambridge, Mass.
