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The uptake and distribution of Co60-labeled vitamin B12 by the fish tapeworm, Diphyllobothrium latum
EXPERIMENTAL
PARASITOLOGY
7,
178-190 (1958)
The Uptake and Distribution BIz by the Fish Tapeworm, Wolmar ZVth Medical University
Clinic,
of CoGO-labeled Vitamin Diphyllobothrium
latum
Nyberg’ Maria
Hospital,
(Submitted for publication,
Helsingfors,
Finland
28 May 1957)
As compared with other living forms, Diphyllobothrium latum contains large amounts of B-12 (Nyberg, 1952). What this means from the standpoint of the metabolism of the parasite is not yet understood. It has not been fully disclosed, either, whether the fish tapeworm satisfies its requirements for B-12 from the intestinal contents, or whether it is also capable of taking up the vitamin directly from its host via the intestinal mucosa. On the basis of previous, preliminary experiments it, seemed probable that B-12 is chiefly taken up from the intestinal contents (Nyberg, 1955). In this communication, which is based on more extensive experiments, a more detailed report on the uptake of vitamin B-12 by the fish tapeworm will be given. MATERIAL
AND METHODS
A total of 126 subjects infected with D. latum, but, not anemic, were investigated. For the sake of comparison, 21 subjects infected with Taenia saginata were also investigated. CoGo-Labeledvitamin B-12 (Merck & Co., specific activity about, 160 pc/mg) was given by mouth in doses varying from 0.34-3.5 pg of B-12 to 118 carriers of D. datum and to 15 carriers of T. saginata. Inorganic Co60
CoGo-Labeledcobalt chloride was administered by mouth to eight subjects harboring the fish tapeworm and to six carriers of T. saginata. The dosagewas calculated so as to contain an amount of cobalt equal to the amount, in 2 pg of B-12. 1 Present Address: Central Hospital of Vasa, Vasa, Finland. 178
UPTAKE
OF
LABELED
B-12
BY
FISH
TAPEWORM
179
Administration of the Labeled Compounds and Expulsion of the Parasites The radioactive vitamin B-12 and the CoGo-labeled cobalt chloride were administered as single doses 12-24 hours before expulsion of the worm. This period was chosen because in preliminary experiments with administration of single doses, the uptake of B-12 in the fish tapeworm seemed to be maximal within this time. Three cases (demonstrated in Table II) have been included in which small daily doses of B-12 were given for 3-6 days to subjects infected with D. latum. The parasites were expelled with Extracturn j&is (dosage 34 g, in some cases of taeniasis up to 7.5 g). Preparation of D. latum Specimens Immediately after expulsion, the worms were repeatedly washed with water. In order to obtain maximal relaxation of the specimens, these were prepared as suggested by Chandler, Read, and Nicholas (1950) in all cases where the whole worm with the scolex intact was obtained. Then the worms were measured and cut into pieces 30-250 cm long, which were separately tested for radioactivity before and after drying. After drying, each piece of worm was weighed. In addition, the total dry weight and the total activity of each worm were checked. Furthermore, in 19 worms the proglottids mere counted, and the radioactivity per proglottid was calculated. In those cases where the worm had come out in pieces, the total mass of worm was dried, weighed and tested for radio-activity. Counting Technic A liquid scintillation counter was used for the measurement of radioactivity. All samples were counted in standard vessels with a constant volume. Background and standard counts were invariably determined before and after counting. Microbiologic
and Chromatographic Checking
During the course of the investigation the radioactive vitamin B-12 was continuously checked microbiologically by its growth-stimulating effect on Lactobacillus leichmannii. In order to check whether the radioactivity taken up by the worm was derived from the radioactive vitamin B-12, an extract of radioactive
180
NYBERG
D. la&m was prepared by a method previously described (Nyberg, 1952). Using butanol-water and lutidine-water as solvents, the radioactivity of this extract was shown chromatographically to be due to B-12. RESULTS
The results from 76 patients are presented in Tables I, II, and III in which the material is classified in three groups corresponding to the dosage of radioactive vitamin B-12: Table I) 0.340.8 pg; Table II) 1.0-1.6 pg and Table III) 2.0-2.68 pg. Both the amount of worm expelled and the uptake of vitamin B-12 in the worm show a wide variation. When the whole material is considered, irrespective of dosage, the uptake in per cent of the dose varies TABLE
I
The Uptake of Coso-Labeled Vitamin B-l.2 in the Fish Tapeworm, D. latum, after Oral Administration oj Radioactive B-f.9 to Subjects Injected with D. latum
Dose B-12,pg.
0.34 0.34 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.6 0.6 0.67 0.67 0.67 0.8
Dry weight, g
12.9 7.8 3.8 11.6 5.2 3.3 19.2 17.8 4.2 7.4 24 6.1 7.8 4.5 9.3 17.6 6.6 10.0 4.0 6.1 6.5 32.2 2.0 4.5
Total Uptake pg B-12 per g dry weight uptake, pg B-12
0.01 0.02 0.034 0.020 0.040 0.021 0.018 0.019 0.035 0.029 0.017 0.019 0.011 0.020 0.036 0.018 0.011 0.010 0.037 0.024 0.056 0.015 0.175 0.695
0.15 0.17 0.13 0.24 0.21 0.07 0.36 0.34 0.15 0.22 0.41 0.12 0.088 0.091 0.34 0.32 0.075 0.10 0.15 0.15 0.37 0.50 0.35 0.43
Uptake, %
45.2 52 27.1 49 43.3 14.1 72.9 68.2 31.3 44.4 83.6 24.1 17.6 18.2 69.7 65.3 15.1 20.4 25 26.6 56.6 75.5 53.7 53.9
UPTAKE
OF LABELED
B-12
BY
FISH
181
TAPEWORM
TABLE II The Uptake of CooaQ-Labeled Vitamin B-18 in the Fish Tapeworm, D. latum, after Oral Administration of Radioactive B-l.3 to Subjects Infected with D. latum
Uptake fig
Dose B-12, cg
Dry weight, g
1.0
13.5 7.7 15 2.2 6.1 22.2 3.7 19
0.0058 0.060 0.047 0.127 0.101 0.024 0.175 0.046
4.1 32.8 6.2
0.064 0.013 0.062
2.2 12.0 1.2 3.3 10.8 10.7 2.5 6.5 20.5 12.2 13.5
0.018 0.067 0.175 0.123 0.067 0.054 0.174 0.106 0.026 0.049 0.072
4.6 2.7 10.4
0.199 0.255 0.067
1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.2 1.2 1.3 1.34 1.34 1.34 1.34 1.34 1.34 1.5 1.5 1.6 1.6
B-12 per g dry weight
Total uptake, pg B-12
Uptake, %
0.788 0.462 0.714 0.280 0.620 0.538 0.650 0.880 0.517 0.264 0.439 0.388 0.232 0.478 0.041 0.808 0.211 0.406 0.724 0.581 0.436 0.691 0.552 0.600 0.978 0.930 0.916 0.691 0.699
78.8 46.2 71.4 28.0 62.0 53.8 65.0 88.0 51.7 26.4 43.9 38.8 23.2 47.8 4.1 80.8 17.6 33.9 55.7 43.4 32.6 51.6 41.2 44.8 73.0 62.0 61.1 43.2 43.7
from 4.1-88. Calculating a) the total uptake and b) the uptake per g of dry worm substance in the whole material, the variation ranges from a) 0.07-1.72 pg and b) 0.01-0.65 pg. In the three groups, the mean values for the uptake as a percentage and for the total uptake are 43.9 % and 0.24 ccg, 48.7 % and 0.57 pg, and 39.3 % and 0.78 pg of B-12. The mean uptake in the whole material is 44.3 % and 0.49 pg. In eight cases of infection with D. latum and six cases of taeniasis in which cobalt chloride was administered, the worms exhibited traces of
182
NYBERG
radioactivity in only two cases (T. saginatu). After thorough washing with water, or following dialysis (for one day at + 4”C.), this radioactivity disappeared. In this connection it should be mentioned that the radioactive vitamin B-12 taken up by D. latum is bound in a non-dialyzable form (Nyberg and Gr&sbeck, 1957). In 15 cases of infection with Tueniu suginutu, vitamin B-12 was administered by mouth in the following doses: 0.5 pg in 2 cases, 1.0 pg in 7 cases, 1.5 pg in one case, and 2.0 pg in 5 cases.After expulsion of the worm, only one specimen (dose 2.0 pg of vitamin B-12) contained significant amounts of radioactivity (1.2 % of the dose or 0.024 pg of B-12). Table IV shows the number of worms per patient expelled from 25 TABLE
III
The Uptake of Coeo-Labeled Vitamin B-12 in the Fish Tapeworm, D. latum, after Oral Administration of Radioactive B-18 to Sub.jects Injected with D. latum
Dose B-12, pg
Dry weight, g.
Uptake pg B-12 per g dry weight
Total uptake, pg B-12
Uptake, %
2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.1 2.68 2.68
3.0 15.8 0.6 8.6 3.1
0.074 0.649 0.260 0.127 0.219
15 16 4.9 8.8 3.5 0.6 4 8.9 3.5 11.0 13.6 11.9 34.8 12.9 4.9 10.6
0.060 0.051 0.184 0.111 0.171 0.390 0.211 0.122 0.177 0.156 0.030 0.066 0.043 0.076 0.290 0.053
0.224 1.026 0.156 1.096 0.680 0.348 0.820 0.904 0.822 0.904 0.980 0.600 0.234 0.844 1.092 0.622 1.722 0.412 0.668 1.516 0.991 1.425 0.565
11.2 51.3 7.8 54.8 34.0 17.4 41.0 45.2 41.1 45.2 49.0 30.0 11.7 42.2 54.6 31.1 86.1 20.6 33.4 75.8 47.2 53.2 21.1
UPTAKE
OF LABELED
B-12
BY FISH TAPEWORM
183 M
No
6 cc
P
6 tic4
2 1
c;
66
8N dd
184
NYBERG
I
2
3
4 Length
5
6 of
7 worm
8
9
IO
II
12
in meters
FIG. 1. Distribution of radioactive B-12 uptake in total counts/minute/piece of the worm in 12 specimens of D. Zatum. Tapeworm Nos. 10, 11 and 12 from the same patient. WOliIl
10 263 3@ 40 50 6a 70 SC3 90 10x 1lA 120
Dose, #g B-12
0.5 0.5 0.5 1.0 1.0 1.0 1.3 2.0 2.5 4.0
Dry
weight g
7.4 3.3 5.2 6.1 2.2 2.6 7.7 4.9 2.8 4.3 3.6 5.8
Length
cm
1210 353 710 958 411 610 624 462 464 700 539 849
Total
uptake
0.22 0.07 0.24 0.62 0.30 0.20 0.72 0.90 1.24 1.46 0.68 0.98
pg B-12
UPTAKE
OF
LABELED
B-12
BY
FISH
185
TAPEWORM
subjects infected with D. latum. It appears that nine subjects (Nos. 17-25) harbored 3, 4, 5, 6, 9, 10, 13, 18 and 20 specimens of the fish tapeworm, respectively. From five patients (Nos. 12-16) two specimens each were expelled, and from 11 subjects only one (Nos. l-11). The uptake of vitamin B-12 in the specimens shows a relatively high degree of dispersion. Particularly in cases with a large number of worms, the supply of B-12 was not equal for all specimens. Figure 1 is a graphical representation of the total activity in various parts of 12 radioactive specimens of the fish tapeworm. In all cases, one (No. 1) excepted, the pattern is the same, the greatest activity occurring at the proximal end of the worm, about 1.5-5 meters from the 17000 16000 15000 14000 13000 12000 E P
“0°0
2
10000
E p 9000 .E 5 8000 L.2 E 3 7000 :: 6000 5000 4000 3000 2000 1000
I
2
3
4 Length
5
6 of
7 worm
8
9
IO
II
I2
13
in meters
FIG. 2. Distribution of radioactive B-12 uptake in counts/minute/g of dry worm/piece of the worm in the 12 specimens of D. latum presented in Fig. 1.
2
3
Length
4
5
of
6
7 worm
FIG. 3. Distribution of radioactive imens of D. .‘atum presented inJ?ig. 1.
I
8
9
IO
B-12 uptake
in meters
Scolex
12
in counts/minute/proglottid
II
2331
95 263 201 154
100
a 608 190 176 164 154 la0 106
X 550 140 123 130 97 90 86 83 76 92 76 125 67 63 67 60 48 44 35 30 31 29 2139
Number a
1565
640 320 300 305
2008
410 300 273 234 207 195 190 200
9
3203
500 528 682 588 409 267 229
in Nos. 3, 4, 6, 10, 11 and 12 of the spec-
2979
552 178 222 190 256 263 420 323 390 185
0
of proglottids
E
UPTAKE
OF
LABELED
B-12
BY
FISH
187
TAPEWORM
scolex, depending on the length of the specimen. The activity curves fall rapidly at the proximal end and relatively slowly at the distal end. When the activity in the various parts was calculated per g of dry worm substance, the curves shown in Fig. 2 were obtained. It appears that the uptake of vitamin B-12 expressed as radioactivity per g of dry weight is definitely greatest in the growth zone of the worm. The uptake of B-12 per proglottid shows the same pattern, broadly speaking, as the distribution of the total activity (cf. Figs. 1 and 3). In Fig. 3, the distribution of the radioactivity per proglottid in the worms Nos. 3, 4, 6, 10, 11 and 12 in Fig. 1 is shown. The remaining casesfor which the uptake of vitamin B-12 per proglottid has been calculated are demonstrated in Fig. 4. I
019JI E0
-
0.34
Scolex H
A740’172.110. 8 0.53 0.23 0.11
*510. 4.4
3.9
t A 808 60.52
248 0.68
B I.5
I Worm A Number of proglottids B Counts/min./progWtid
I
1 : Ei 2
164 279 1.06 0.67
t : A420 372 80.5 4.75
k ;
As% I
Strobila -
:.:
0.3 206
3 I
311 2.36
312 0.87 I
382 3.1
501 3.3
‘d?s”
:
kT2 E
t : A522 393 80.4 I.9 F : A550 400 80.34 2.62 A%%783765 I” :
1 223 I.17
0.34 179
0.27 190 4
553 1.4
261 0.4 I
‘2; : 309
250
I89 0.65 226
“I?? 235
I
6%
80.2 0.75 I.16 I.31 I.14 0.99 0.93 0.68 I : A513 370 7% E.: :.I? I96 190 174 SO.29 I.11 0.96 1.48 1.07 I : A440 455 427 404 346 80.46 3.06 2.87 I.19 0.81
I 0
100
200
Length
300
of
FIG. 4. Distribution of radioactive in 13 specimens of D. latum.
400
worm
500
in
600
700
191 0.57
I i
279 0.53
800
I 900
centimeters
B-12 uptake
in counts/minute/proglottid
188
NYBERG DISCUSSION
It appears that the fish tapeworm, in contrast to Taenia saginata, had taken up radioactive vitamin B-12 in all investigated cases. The amount absorbed after administration of a single dose varied greatly in the individual cases. Although the variation was to some extent dependent on the dosage, the magnitude of the uptake seemsto depend on a variety of unknown factors. Evidently the location of the worm in the intestine plays a significant part. In view of the fact that the absorption of the vitamin occurs chiefly in the proximal part of the intestine it would seem that worms located in the duodenum or in the proximal parts of the jejunum are in a much better position, from the standpoint of the supply of vitamin B-12, than worms in more distal sites. This assumption is substantiated by the finding that in casesof fish tapeworm anemia D. latum absorbs larger amounts of the radioactive vitamin than in casesof tapeworm infection without anemia. According to von Bonsdorff it is probable that the worm is situated high up in the small intestine in cases of fish tapeworm anemia (for discussion and references seevon Bonsdorff, 1956). It seems possible that the stage of sexual maturity of the worm at the time of investigation is another important factor. In dogs, Wardle and Green (1941) found that the weight/length ratio of D. Zatum rapidly increased up to the 18th day after infection. Subsequently a considerable drop was observed, which coincided with the first discharge of eggs. Then the weight/length ratio again rose abruptly. After the discharge of eggs the proglottids have been found to lose from one third to half of their weight. Every factor that stimulates egg production tends to increase the weight/length ratio, whilst substances inhibiting egg production stimulate the linear growth with a low weight/length ratio resulting. In this connection the uneven distribution of the radioactivity in the expelled worms is of interest, the activity being greatest in the proximal part of the worm both when calculated as total uptake and as uptake per proglottid or per g of dry substance. The maximum of the total uptake was observed about 1.5-5 meters from the scolex depending on the length of the worm. At the proximal end the decline in total uptake was much more rapid than distally. This concentration of vitamin B-12 in the growth zone and the developing proglottids of the worm seems to indicate that a phenomenon is involved other than simple diffusion resulting in an exchange of the vitamin between the worm tissue and the surrounding intestinal contents. The fact
UPTAKE OF LABELED B-12 BY FISH TAPEWORM
189
that inorganic cobalt is not absorbed by D. latum also constitutes evidence in favor of the assumption that the uptake of vitamin B-12 is specific. It is conceivable that this vitamin may be a growth- or maturation-promoting factor for D. latum. Furthermore, the uptake of vitamin B-12 by the worm may be influenced by the bacterial flora. At least theoretically it may be assumed that the latter competes with the parasite for the vitamin. In addition, various nutritional factors may play a part. Finally it should be borne in mind that it is not always possible to decide immediately after a worm cure whether all of the parasite has been expehed. Hence check-up examinations for the presence of tapeworm were performed from 3 to 8 weeks later in some 10 cases. In four of these, tapeworm eggs were observed in the feces. Considering, however, that the possibility of reinfection cannot be eliminated in a population whose diet includes fresh-water fish as a main ingredient and who regard raw fish as a delicacy, the value of such a check-up examination is not significant. It should also be emphasized that even a small dose of vitamin B-12 is not entirely absorbed from the intestine, and that probably the uptake by D. latum is not complete, either. This seems to be at least in part due to the occurence of non-absorbable breakdown products of the radioactive vitamin B-12 (Mollin and Baker, 1955). SUMMARY
The fish tapeworm, Diphyllobothrium latum, absorbs an average of about 44 % of a single oral dose of radioactive vitamin B-12 given to the host. The individual variations are great and seem to be due to a variety of unpredictable factors. The radioactivity is mainly concentrated in the proximal part of the worm. Taenia saginata does not take up any radioactive vitamin B-12 when the host has been given a single dose by mouth. CoGo-Labeled cobalt chloride orally administered to patients infected with D. latum or T. saginata is not absorbed by the parasites. ACKNOWLEDGMENTS I wish to thank Dr. H. Renkonen, and Dr. T. Kalaja, for permitting me to collect the material and perform a major part of the work at the Joensuu Central Hospital. I am also indebted to Messrs. Merck & Co., who placed radioactive vitamin B-12 at my disposal. This investigation has been supported by a grant from the Sigrid Juselius Stiftelse.
190
NYBERG REFERENCES
VON BONSDORFF, B. 1956. Diphyllobothrium latum as a cause of pernicious anemia. In Parasitological Reviews Section of Ezptl. Parasitol. 6, 207-230. CHANDLER, A. C., READ, C. P., AND NICHOLAS, H. 0.1950. Observations on certain phases of nutrition and host-parasite relations of Hymenolepis diminuta in white rats. J. Parasitol. 36, 533-535. MOLLIN, D. L., AND BAKER, S. J. 1955. The absorption and excretion of vitamin B-12 in man. Biochem. Sot. Symposia (Cambridge, Engl.) 13, 52-68. NYBERG, W. 1952. Microbiological investigations on antipernicious anemia factors in the fish tapeworm. Acta med. stand. suppl. 271. NYBERG, W. 1956. Uptake of CoGO-labeled vitamin B-12 in the fish tapeworm. V
Kongress der Europaischen Gesellschaft fur Hiimatologie. Springer-Verlag, Berlin-Gottingen-Heidelberg, p. 58-60. NYBERG, W., AND GRXSBECK, R. 1957. Is VitaminB-12 in bound or free form in the tapeworm Diphyllobothrium latum? &and. J. Clin. Lab. Invest., 9, 383-387. WARDLE, R. A., AND GREEN, N. K. 1941. quoted by WARDLE, R. A., AND MCLEOD, I. A. 1952. The Zoology of Tapeworms. The University Press, Minneapolis.
PARASITOLOGY
7,
178-190 (1958)
The Uptake and Distribution BIz by the Fish Tapeworm, Wolmar ZVth Medical University
Clinic,
of CoGO-labeled Vitamin Diphyllobothrium
latum
Nyberg’ Maria
Hospital,
(Submitted for publication,
Helsingfors,
Finland
28 May 1957)
As compared with other living forms, Diphyllobothrium latum contains large amounts of B-12 (Nyberg, 1952). What this means from the standpoint of the metabolism of the parasite is not yet understood. It has not been fully disclosed, either, whether the fish tapeworm satisfies its requirements for B-12 from the intestinal contents, or whether it is also capable of taking up the vitamin directly from its host via the intestinal mucosa. On the basis of previous, preliminary experiments it, seemed probable that B-12 is chiefly taken up from the intestinal contents (Nyberg, 1955). In this communication, which is based on more extensive experiments, a more detailed report on the uptake of vitamin B-12 by the fish tapeworm will be given. MATERIAL
AND METHODS
A total of 126 subjects infected with D. latum, but, not anemic, were investigated. For the sake of comparison, 21 subjects infected with Taenia saginata were also investigated. CoGo-Labeledvitamin B-12 (Merck & Co., specific activity about, 160 pc/mg) was given by mouth in doses varying from 0.34-3.5 pg of B-12 to 118 carriers of D. datum and to 15 carriers of T. saginata. Inorganic Co60
CoGo-Labeledcobalt chloride was administered by mouth to eight subjects harboring the fish tapeworm and to six carriers of T. saginata. The dosagewas calculated so as to contain an amount of cobalt equal to the amount, in 2 pg of B-12. 1 Present Address: Central Hospital of Vasa, Vasa, Finland. 178
UPTAKE
OF
LABELED
B-12
BY
FISH
TAPEWORM
179
Administration of the Labeled Compounds and Expulsion of the Parasites The radioactive vitamin B-12 and the CoGo-labeled cobalt chloride were administered as single doses 12-24 hours before expulsion of the worm. This period was chosen because in preliminary experiments with administration of single doses, the uptake of B-12 in the fish tapeworm seemed to be maximal within this time. Three cases (demonstrated in Table II) have been included in which small daily doses of B-12 were given for 3-6 days to subjects infected with D. latum. The parasites were expelled with Extracturn j&is (dosage 34 g, in some cases of taeniasis up to 7.5 g). Preparation of D. latum Specimens Immediately after expulsion, the worms were repeatedly washed with water. In order to obtain maximal relaxation of the specimens, these were prepared as suggested by Chandler, Read, and Nicholas (1950) in all cases where the whole worm with the scolex intact was obtained. Then the worms were measured and cut into pieces 30-250 cm long, which were separately tested for radioactivity before and after drying. After drying, each piece of worm was weighed. In addition, the total dry weight and the total activity of each worm were checked. Furthermore, in 19 worms the proglottids mere counted, and the radioactivity per proglottid was calculated. In those cases where the worm had come out in pieces, the total mass of worm was dried, weighed and tested for radio-activity. Counting Technic A liquid scintillation counter was used for the measurement of radioactivity. All samples were counted in standard vessels with a constant volume. Background and standard counts were invariably determined before and after counting. Microbiologic
and Chromatographic Checking
During the course of the investigation the radioactive vitamin B-12 was continuously checked microbiologically by its growth-stimulating effect on Lactobacillus leichmannii. In order to check whether the radioactivity taken up by the worm was derived from the radioactive vitamin B-12, an extract of radioactive
180
NYBERG
D. la&m was prepared by a method previously described (Nyberg, 1952). Using butanol-water and lutidine-water as solvents, the radioactivity of this extract was shown chromatographically to be due to B-12. RESULTS
The results from 76 patients are presented in Tables I, II, and III in which the material is classified in three groups corresponding to the dosage of radioactive vitamin B-12: Table I) 0.340.8 pg; Table II) 1.0-1.6 pg and Table III) 2.0-2.68 pg. Both the amount of worm expelled and the uptake of vitamin B-12 in the worm show a wide variation. When the whole material is considered, irrespective of dosage, the uptake in per cent of the dose varies TABLE
I
The Uptake of Coso-Labeled Vitamin B-l.2 in the Fish Tapeworm, D. latum, after Oral Administration oj Radioactive B-f.9 to Subjects Injected with D. latum
Dose B-12,pg.
0.34 0.34 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.6 0.6 0.67 0.67 0.67 0.8
Dry weight, g
12.9 7.8 3.8 11.6 5.2 3.3 19.2 17.8 4.2 7.4 24 6.1 7.8 4.5 9.3 17.6 6.6 10.0 4.0 6.1 6.5 32.2 2.0 4.5
Total Uptake pg B-12 per g dry weight uptake, pg B-12
0.01 0.02 0.034 0.020 0.040 0.021 0.018 0.019 0.035 0.029 0.017 0.019 0.011 0.020 0.036 0.018 0.011 0.010 0.037 0.024 0.056 0.015 0.175 0.695
0.15 0.17 0.13 0.24 0.21 0.07 0.36 0.34 0.15 0.22 0.41 0.12 0.088 0.091 0.34 0.32 0.075 0.10 0.15 0.15 0.37 0.50 0.35 0.43
Uptake, %
45.2 52 27.1 49 43.3 14.1 72.9 68.2 31.3 44.4 83.6 24.1 17.6 18.2 69.7 65.3 15.1 20.4 25 26.6 56.6 75.5 53.7 53.9
UPTAKE
OF LABELED
B-12
BY
FISH
181
TAPEWORM
TABLE II The Uptake of CooaQ-Labeled Vitamin B-18 in the Fish Tapeworm, D. latum, after Oral Administration of Radioactive B-l.3 to Subjects Infected with D. latum
Uptake fig
Dose B-12, cg
Dry weight, g
1.0
13.5 7.7 15 2.2 6.1 22.2 3.7 19
0.0058 0.060 0.047 0.127 0.101 0.024 0.175 0.046
4.1 32.8 6.2
0.064 0.013 0.062
2.2 12.0 1.2 3.3 10.8 10.7 2.5 6.5 20.5 12.2 13.5
0.018 0.067 0.175 0.123 0.067 0.054 0.174 0.106 0.026 0.049 0.072
4.6 2.7 10.4
0.199 0.255 0.067
1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.2 1.2 1.3 1.34 1.34 1.34 1.34 1.34 1.34 1.5 1.5 1.6 1.6
B-12 per g dry weight
Total uptake, pg B-12
Uptake, %
0.788 0.462 0.714 0.280 0.620 0.538 0.650 0.880 0.517 0.264 0.439 0.388 0.232 0.478 0.041 0.808 0.211 0.406 0.724 0.581 0.436 0.691 0.552 0.600 0.978 0.930 0.916 0.691 0.699
78.8 46.2 71.4 28.0 62.0 53.8 65.0 88.0 51.7 26.4 43.9 38.8 23.2 47.8 4.1 80.8 17.6 33.9 55.7 43.4 32.6 51.6 41.2 44.8 73.0 62.0 61.1 43.2 43.7
from 4.1-88. Calculating a) the total uptake and b) the uptake per g of dry worm substance in the whole material, the variation ranges from a) 0.07-1.72 pg and b) 0.01-0.65 pg. In the three groups, the mean values for the uptake as a percentage and for the total uptake are 43.9 % and 0.24 ccg, 48.7 % and 0.57 pg, and 39.3 % and 0.78 pg of B-12. The mean uptake in the whole material is 44.3 % and 0.49 pg. In eight cases of infection with D. latum and six cases of taeniasis in which cobalt chloride was administered, the worms exhibited traces of
182
NYBERG
radioactivity in only two cases (T. saginatu). After thorough washing with water, or following dialysis (for one day at + 4”C.), this radioactivity disappeared. In this connection it should be mentioned that the radioactive vitamin B-12 taken up by D. latum is bound in a non-dialyzable form (Nyberg and Gr&sbeck, 1957). In 15 cases of infection with Tueniu suginutu, vitamin B-12 was administered by mouth in the following doses: 0.5 pg in 2 cases, 1.0 pg in 7 cases, 1.5 pg in one case, and 2.0 pg in 5 cases.After expulsion of the worm, only one specimen (dose 2.0 pg of vitamin B-12) contained significant amounts of radioactivity (1.2 % of the dose or 0.024 pg of B-12). Table IV shows the number of worms per patient expelled from 25 TABLE
III
The Uptake of Coeo-Labeled Vitamin B-12 in the Fish Tapeworm, D. latum, after Oral Administration of Radioactive B-18 to Sub.jects Injected with D. latum
Dose B-12, pg
Dry weight, g.
Uptake pg B-12 per g dry weight
Total uptake, pg B-12
Uptake, %
2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.1 2.68 2.68
3.0 15.8 0.6 8.6 3.1
0.074 0.649 0.260 0.127 0.219
15 16 4.9 8.8 3.5 0.6 4 8.9 3.5 11.0 13.6 11.9 34.8 12.9 4.9 10.6
0.060 0.051 0.184 0.111 0.171 0.390 0.211 0.122 0.177 0.156 0.030 0.066 0.043 0.076 0.290 0.053
0.224 1.026 0.156 1.096 0.680 0.348 0.820 0.904 0.822 0.904 0.980 0.600 0.234 0.844 1.092 0.622 1.722 0.412 0.668 1.516 0.991 1.425 0.565
11.2 51.3 7.8 54.8 34.0 17.4 41.0 45.2 41.1 45.2 49.0 30.0 11.7 42.2 54.6 31.1 86.1 20.6 33.4 75.8 47.2 53.2 21.1
UPTAKE
OF LABELED
B-12
BY FISH TAPEWORM
183 M
No
6 cc
P
6 tic4
2 1
c;
66
8N dd
184
NYBERG
I
2
3
4 Length
5
6 of
7 worm
8
9
IO
II
12
in meters
FIG. 1. Distribution of radioactive B-12 uptake in total counts/minute/piece of the worm in 12 specimens of D. Zatum. Tapeworm Nos. 10, 11 and 12 from the same patient. WOliIl
10 263 3@ 40 50 6a 70 SC3 90 10x 1lA 120
Dose, #g B-12
0.5 0.5 0.5 1.0 1.0 1.0 1.3 2.0 2.5 4.0
Dry
weight g
7.4 3.3 5.2 6.1 2.2 2.6 7.7 4.9 2.8 4.3 3.6 5.8
Length
cm
1210 353 710 958 411 610 624 462 464 700 539 849
Total
uptake
0.22 0.07 0.24 0.62 0.30 0.20 0.72 0.90 1.24 1.46 0.68 0.98
pg B-12
UPTAKE
OF
LABELED
B-12
BY
FISH
185
TAPEWORM
subjects infected with D. latum. It appears that nine subjects (Nos. 17-25) harbored 3, 4, 5, 6, 9, 10, 13, 18 and 20 specimens of the fish tapeworm, respectively. From five patients (Nos. 12-16) two specimens each were expelled, and from 11 subjects only one (Nos. l-11). The uptake of vitamin B-12 in the specimens shows a relatively high degree of dispersion. Particularly in cases with a large number of worms, the supply of B-12 was not equal for all specimens. Figure 1 is a graphical representation of the total activity in various parts of 12 radioactive specimens of the fish tapeworm. In all cases, one (No. 1) excepted, the pattern is the same, the greatest activity occurring at the proximal end of the worm, about 1.5-5 meters from the 17000 16000 15000 14000 13000 12000 E P
“0°0
2
10000
E p 9000 .E 5 8000 L.2 E 3 7000 :: 6000 5000 4000 3000 2000 1000
I
2
3
4 Length
5
6 of
7 worm
8
9
IO
II
I2
13
in meters
FIG. 2. Distribution of radioactive B-12 uptake in counts/minute/g of dry worm/piece of the worm in the 12 specimens of D. latum presented in Fig. 1.
2
3
Length
4
5
of
6
7 worm
FIG. 3. Distribution of radioactive imens of D. .‘atum presented inJ?ig. 1.
I
8
9
IO
B-12 uptake
in meters
Scolex
12
in counts/minute/proglottid
II
2331
95 263 201 154
100
a 608 190 176 164 154 la0 106
X 550 140 123 130 97 90 86 83 76 92 76 125 67 63 67 60 48 44 35 30 31 29 2139
Number a
1565
640 320 300 305
2008
410 300 273 234 207 195 190 200
9
3203
500 528 682 588 409 267 229
in Nos. 3, 4, 6, 10, 11 and 12 of the spec-
2979
552 178 222 190 256 263 420 323 390 185
0
of proglottids
E
UPTAKE
OF
LABELED
B-12
BY
FISH
187
TAPEWORM
scolex, depending on the length of the specimen. The activity curves fall rapidly at the proximal end and relatively slowly at the distal end. When the activity in the various parts was calculated per g of dry worm substance, the curves shown in Fig. 2 were obtained. It appears that the uptake of vitamin B-12 expressed as radioactivity per g of dry weight is definitely greatest in the growth zone of the worm. The uptake of B-12 per proglottid shows the same pattern, broadly speaking, as the distribution of the total activity (cf. Figs. 1 and 3). In Fig. 3, the distribution of the radioactivity per proglottid in the worms Nos. 3, 4, 6, 10, 11 and 12 in Fig. 1 is shown. The remaining casesfor which the uptake of vitamin B-12 per proglottid has been calculated are demonstrated in Fig. 4. I
019JI E0
-
0.34
Scolex H
A740’172.110. 8 0.53 0.23 0.11
*510. 4.4
3.9
t A 808 60.52
248 0.68
B I.5
I Worm A Number of proglottids B Counts/min./progWtid
I
1 : Ei 2
164 279 1.06 0.67
t : A420 372 80.5 4.75
k ;
As% I
Strobila -
:.:
0.3 206
3 I
311 2.36
312 0.87 I
382 3.1
501 3.3
‘d?s”
:
kT2 E
t : A522 393 80.4 I.9 F : A550 400 80.34 2.62 A%%783765 I” :
1 223 I.17
0.34 179
0.27 190 4
553 1.4
261 0.4 I
‘2; : 309
250
I89 0.65 226
“I?? 235
I
6%
80.2 0.75 I.16 I.31 I.14 0.99 0.93 0.68 I : A513 370 7% E.: :.I? I96 190 174 SO.29 I.11 0.96 1.48 1.07 I : A440 455 427 404 346 80.46 3.06 2.87 I.19 0.81
I 0
100
200
Length
300
of
FIG. 4. Distribution of radioactive in 13 specimens of D. latum.
400
worm
500
in
600
700
191 0.57
I i
279 0.53
800
I 900
centimeters
B-12 uptake
in counts/minute/proglottid
188
NYBERG DISCUSSION
It appears that the fish tapeworm, in contrast to Taenia saginata, had taken up radioactive vitamin B-12 in all investigated cases. The amount absorbed after administration of a single dose varied greatly in the individual cases. Although the variation was to some extent dependent on the dosage, the magnitude of the uptake seemsto depend on a variety of unknown factors. Evidently the location of the worm in the intestine plays a significant part. In view of the fact that the absorption of the vitamin occurs chiefly in the proximal part of the intestine it would seem that worms located in the duodenum or in the proximal parts of the jejunum are in a much better position, from the standpoint of the supply of vitamin B-12, than worms in more distal sites. This assumption is substantiated by the finding that in casesof fish tapeworm anemia D. latum absorbs larger amounts of the radioactive vitamin than in casesof tapeworm infection without anemia. According to von Bonsdorff it is probable that the worm is situated high up in the small intestine in cases of fish tapeworm anemia (for discussion and references seevon Bonsdorff, 1956). It seems possible that the stage of sexual maturity of the worm at the time of investigation is another important factor. In dogs, Wardle and Green (1941) found that the weight/length ratio of D. Zatum rapidly increased up to the 18th day after infection. Subsequently a considerable drop was observed, which coincided with the first discharge of eggs. Then the weight/length ratio again rose abruptly. After the discharge of eggs the proglottids have been found to lose from one third to half of their weight. Every factor that stimulates egg production tends to increase the weight/length ratio, whilst substances inhibiting egg production stimulate the linear growth with a low weight/length ratio resulting. In this connection the uneven distribution of the radioactivity in the expelled worms is of interest, the activity being greatest in the proximal part of the worm both when calculated as total uptake and as uptake per proglottid or per g of dry substance. The maximum of the total uptake was observed about 1.5-5 meters from the scolex depending on the length of the worm. At the proximal end the decline in total uptake was much more rapid than distally. This concentration of vitamin B-12 in the growth zone and the developing proglottids of the worm seems to indicate that a phenomenon is involved other than simple diffusion resulting in an exchange of the vitamin between the worm tissue and the surrounding intestinal contents. The fact
UPTAKE OF LABELED B-12 BY FISH TAPEWORM
189
that inorganic cobalt is not absorbed by D. latum also constitutes evidence in favor of the assumption that the uptake of vitamin B-12 is specific. It is conceivable that this vitamin may be a growth- or maturation-promoting factor for D. latum. Furthermore, the uptake of vitamin B-12 by the worm may be influenced by the bacterial flora. At least theoretically it may be assumed that the latter competes with the parasite for the vitamin. In addition, various nutritional factors may play a part. Finally it should be borne in mind that it is not always possible to decide immediately after a worm cure whether all of the parasite has been expehed. Hence check-up examinations for the presence of tapeworm were performed from 3 to 8 weeks later in some 10 cases. In four of these, tapeworm eggs were observed in the feces. Considering, however, that the possibility of reinfection cannot be eliminated in a population whose diet includes fresh-water fish as a main ingredient and who regard raw fish as a delicacy, the value of such a check-up examination is not significant. It should also be emphasized that even a small dose of vitamin B-12 is not entirely absorbed from the intestine, and that probably the uptake by D. latum is not complete, either. This seems to be at least in part due to the occurence of non-absorbable breakdown products of the radioactive vitamin B-12 (Mollin and Baker, 1955). SUMMARY
The fish tapeworm, Diphyllobothrium latum, absorbs an average of about 44 % of a single oral dose of radioactive vitamin B-12 given to the host. The individual variations are great and seem to be due to a variety of unpredictable factors. The radioactivity is mainly concentrated in the proximal part of the worm. Taenia saginata does not take up any radioactive vitamin B-12 when the host has been given a single dose by mouth. CoGo-Labeled cobalt chloride orally administered to patients infected with D. latum or T. saginata is not absorbed by the parasites. ACKNOWLEDGMENTS I wish to thank Dr. H. Renkonen, and Dr. T. Kalaja, for permitting me to collect the material and perform a major part of the work at the Joensuu Central Hospital. I am also indebted to Messrs. Merck & Co., who placed radioactive vitamin B-12 at my disposal. This investigation has been supported by a grant from the Sigrid Juselius Stiftelse.
190
NYBERG REFERENCES
VON BONSDORFF, B. 1956. Diphyllobothrium latum as a cause of pernicious anemia. In Parasitological Reviews Section of Ezptl. Parasitol. 6, 207-230. CHANDLER, A. C., READ, C. P., AND NICHOLAS, H. 0.1950. Observations on certain phases of nutrition and host-parasite relations of Hymenolepis diminuta in white rats. J. Parasitol. 36, 533-535. MOLLIN, D. L., AND BAKER, S. J. 1955. The absorption and excretion of vitamin B-12 in man. Biochem. Sot. Symposia (Cambridge, Engl.) 13, 52-68. NYBERG, W. 1952. Microbiological investigations on antipernicious anemia factors in the fish tapeworm. Acta med. stand. suppl. 271. NYBERG, W. 1956. Uptake of CoGO-labeled vitamin B-12 in the fish tapeworm. V
Kongress der Europaischen Gesellschaft fur Hiimatologie. Springer-Verlag, Berlin-Gottingen-Heidelberg, p. 58-60. NYBERG, W., AND GRXSBECK, R. 1957. Is VitaminB-12 in bound or free form in the tapeworm Diphyllobothrium latum? &and. J. Clin. Lab. Invest., 9, 383-387. WARDLE, R. A., AND GREEN, N. K. 1941. quoted by WARDLE, R. A., AND MCLEOD, I. A. 1952. The Zoology of Tapeworms. The University Press, Minneapolis.