Flame photometric studies of helminths: Calcium, magnesium, potassium, and sodium in Hymenolepis diminuta

EXPERL,NIENTAL PARASITOLOGy 12, 107-113

(1962)

F l a m e P h o t o m e t r i c Studies of H e l m i n t h s : Calcium, Magnesium, Potassium, and S o d i u m in Hymenolepis diminuta 1

Chauncey G. Goodehild, Elizabeth S. Dennis, and John D. Moore Department o] Biology, Emory University, Atlanta, Georgia (Submitted for publication, 11 September 1961) 1. The cations Ca, Mg, K, and Na in freeze-dried Hymenolepis diminuta have been determined by emission spectroscopy. 2. In anterior, middle, and posterior thirds of dried worms percentages of Ca were 0.097 • .010, 0.086 -4- .007, 0.061 • .004; of Mg were 0.063 • .002, 0.058~ .002, 0.064• .001 ; of K were 0.896 ~ .022, 0.869 -4- .040, 0.771 ~ .025 ; and percentages of Na in the same thirds were 0.260 • .016, 0.318 -4- .017, 0.345 • .018. 3. The sum of percentages of the four cations in dried anterior thirds was 1.32 • .04, in middle thirds was 1.33 • .05, and in posterior thirds was 1.24 • .04. 4. In absolute amounts (percent X dry weight, in rag), the cations in anterior, middle, and posterior thirds, respectively, were for Ca 0.019, 0.043, 0.031; for Mg were 0.013, 0.029, 0.033; for K were 0.190, 0.446, 0.390; and for Na were 0.056, 0.164, 0.177. Whole dried worms contained 0.092 mg (0.075%) of Ca, 0.075 mg (0.061%) of Mg, 1.027mg (0.836%) of K, and 0.397 mg (0.323%) of Na. The composite absolute amount of these four cations in dried H. diminuta was 1.591 mg, or 1.296%. 5. Dried H. diminuta, like dried host liver and mammalian red blood cells (but unlike serum), had similar quantitative excesses of K over Na and Mg; however, Ca although occurring in lesser amounts than K, was not present in similar K:Ca ratios in tapeworms and host tissues. Recent physiological studies of parasites have dealt extensively with metabolism and metabolic products (von Brand, 1957), for the most part with carbohydrates, lipids, and proteins. Few references are made to the presence of inorganic substances, either qualitatively or quantitatively. T h e nematode Ascaris lumbricoides has been the subject of the most intensive investigations on inorganic contents of helminths. F l u r y (1912) determined that the dry ash of Ascaris constituted 5.07% of the dry weight and 0.75% of the fresh weight and reported inorganic components, based upon percent of dry weight, as follows: Na, 1.104; K, 0.607; 1 This study was supported (in part) by a training grant (PHS-2E-37) from the Institute of Allergy and Infectious Disease, of the National Institutes of Health, U.S. Public Health Service.

Ca, 0.404; and Mg, 0.058; he also quantitatively determined the following: A1, Fe, C1, H~PO4, H2SO4, and H2SiO3. The mineral content of the acanthocephalan Macracanthorhynckus hirudinaceus has been investigated b y von Brand (1939); although no quantitative data were given for identified Na, K, Mg, Ca, Fe, C1, PO4, and SO4, it was determined that inorganic materials comprised 5.0% of the dry weight, or 0.58% of the wet weight. Later, von Brand and Saurwein (1942) added Mn, A1, and Cu to the list of elements known to be present in this acanthocephalan. Inorganic constituents in cystic fluids of larval cestodes (reviewed b y Wardle and McLeod, 1952) and dried larvae and adults have been investigated b y several workers. Elements in larval fluids are not considered 107

108

GOODCHILD, DENNIS, AND MOORE

in this paper because the content of some cysts consists, in part, of a transudate or ultrafiltrate of host serum (Schopfer, 1932; Goodchild and Kagan, 1961); elements present are probably in dynamic balance with host serum and may not be present in equivalent amounts within the tissues of the larva. Salisbury and Anderson (1939) analyzed the dried, fat-free strobilocercus of Hydatigera taeniae]ormis and found that the ash comprised 16.3% of the dry weight and contained the following percentages of elements: Ca, 29.50; Mg, 20.14; K, 3.66; Ha, 6.99; S, 0.74; and P, 5.37. Calcareous corpuscles have long been recognized in larval and adult cestodes; these have recently been studied by von Brand, et al. (1960) in Hydatigera taeniaeJormis, in which they comprised 6.93% and 3.15% of larval and adult wet weight, respectively. Quantitative analysis yielded, in percent of larval wet weight, Ca 1.23, Mg 0.928, P 0.121, and CO2 2.21, and for adult wet weight, Ca 0.630, Mg 0.331, P 0.155, and CO2 0.879. Since so little information is available on the inorganic composition of parasites, it was felt that a quantitative study of the cations Ca, Mg, K, and Na present in adult Hymenolepis diminuta would be beneficial in supplementing other studies. Flame photometry was chosen as an especially applicable method for this analysis. MATERIALS AND METHODS

Five cysticercoids of Hymenolepis diminuta, obtained by dissecting infected Tribolium con]usum in saline, were given by stomach tube to each of eight white rats of the Wistar strain. All rats were maintained on a standard laboratory food supplied ad libitum. Twentyeight days after infection the rats were sacrificed, and each of the 35 tapeworms recovered was placed on a paper towel, blotted to remove adherent water, measured, divided into anterior, middle, and posterior thirds by length, and weighed on a torsion balance. Worms recovered from each rat were combined, by regions, into three weighed bottles, homogenized in distilled water using a microattachment for the Servall Omni-Mixer, and stored in a freezer. Each sample was freezedried, within a few days, the dry weight determined, and was then returned to the freezer

until chemical determinations could be performed. Freeze-dried tapeworms were stored in uncapped bottles over dehydrated silica gel overnight immediately prior to initiation of chemical determinations. Weighed samples (15 to 20 mg) were placed in individual 10 ml Erlenmeyer flasks along with 2 ml of a 5:1 (v:v) mixture of HNO3 (sp gr 1.4) and 70% HC10, (sp gr 1.6). Each flask was covered with a glass marble and placed on a hot plate, the temperature of which was elevated gradually to 1200 C and the contents digested until fumes of NO2 had dissipated and the digest was colorless or pale yellow in color. The samples were uncovered, transferred to a sand bath, tilted approximately 45 ~ and the temperature elevated and held at 210~ until dense white fumes of HC104 were given off. Digests were cooled and transferred quantitatively with de-ionized water to 6-ml hematocrit tubes and diluted to 5 ml for Mg, K, and Na determinations. For the determination of Ca, 1 ml aliquots of the diluted solutions were transferred to test tubes and 0.04 ml KH2PO, solution (1 mg P / m l ) was added to each tube (giving a dilution factor of 5.2). Prior to determination of elements in samples the peaks of maximum emissions, at the selected resonance wave lengths, were determined with the most concentrated standards and the sensitivity dial of the monochromator then adjusted to give 100% T with each standard (Table I ) . Emission of a series of four to six dilutions of standards (ranging in btg/ml down to 0.125 for Ca, to 1.6 for Mg, to 2.5 for K, and to 1.6 for Na) was determined and used to construct graphs from which concentrations of elements in samples were read. In every case experimental values fell within the range of standard values. Samples for Ca determinations were read against Ca3(PO4)2 standards, as suggested by Leyton (1954). The dilution prepared for Mg, K, and Na determinations was read, respectively, against standards of magnesium metal dissolved in a small quantity of 6 N HC1 and diluted with water, against KC1 in water, and against NaC1 in water. Weighed samples of CaCO3, Ca3(PO,)2, MgCO3, NaC1, and KC1 were digested and treated in a manner identical to that used with

109

FLAME PHOTOMETRIC STUDIES OF HELMINTHS

TABLE I Settings on Flame Photometer (Beckman No. 9200) In all determinations selector switch on 0.1, zero suppression on 2, oxygen 18psi, and hydrogen 5psi (each determined by orifices of burner (Beckman No. 4020)). Photomultiplier tube (Beckman No. 4300) with 22 megohm resistor for Ca, Mg, and Na and red-sensitive phototube (Beckman No. 157) with 10,000 megohm resistor for K. Wave length, m~ (resonance line) Background, ml~ ~g/ml for 100% T a Standards Slit width, mm Sensitivity switch on power supply Sensitivity control dialb on monochromator

Calcium

Magnesium

Potassium

Sodium

422.7 418 6.25 Ca3 (PO4) 2 0.05

285.2 280 20 Mg 0.07

769 795 100 KC1 0.065

589 580 40 NaC1 0.03

9

8

Off

5

760

950

940

240

a ~g/ml of element, not compound (0.38750)< Ca3(PO4)2; 0.52443 )< KC1; 0.39336 X NaC1). b This value is approximate and will change with duration of operation of instrument.

tapeworms and the q u a n t i t y of Ca, Mg, K, and N a was determined b y emission spectroscopy. T h e amount of element actually present divided b y the amount found in each determination gave a factor b y which the amounts of each element found in the tapeworm samples were multiplied. A spectrophotometer 2 with flame photometry and photomultiplier attachments, and power supply was used in this work. T h e flame source was a commercial burner 3 using hydrogen and oxygen purchased from a supplier. 4 Emission measurements were made at wave lengths and instrument settings as shown in T a b l e I. T h e original sensitivity control knob on the monochromator, which activates a 10-turn helical potentiometer, was replaced b y a dial calibrated from 0 to 100 over 360 ~ the clockwise limit of which was read as 0 and the counterclockwise limit as 1,000. All emission measurements were m a d e b y the method of b a c k g r o u n d correction as discussed b y Dinnin (1960). T h e y were obtained b y making measurements at two wave lengths, one the resonance line of the element and the second (the b a c k g r o u n d emission) an a d j a c e n t 2 Beckman Model DU: attachments--flame photometry No. 9200, photomultiplier No. 4300; and power supply No. 23700. a Beckman No. 4020. 4 Southern Oxygen Supply Co., Atlanta, Ga.

wave length which gave, while the s t a n d a r d solution was aspirated, an emission equal to or approaching that of de-ionized water. T h e background emission was subtracted from the gross emission of the resonance line to yield a net emission due to the element alone. I n addition, reagent blanks digested and treated identically to the samples were read at resonance and background settings. D a t a were analyzed statistically using " S t u d e n t ' s " t-test for p r o b a b i l i t y of significance of difference between means. RESULTS

Quantitative findings for percentages of Ca, Mg, K, and N a , and a percentage total of these four cations in normal, freeze-dried Hymenolepis diminuta are shown in T a b l e I I and in Figs. 1 and 2. Results are presented for anterior, middle, and posterior thirds. "P" values at the 5% level are considered to represent significant differences. T o t a l Ca (percent X d r y weight) from anterior, middle, and posterior regions were 0.019, 0.043, and 0.031 mg, respectively; in whole tapeworms (average d r y weight, 122.8 mg) Ca totalled 0.092 mg, or 0.075% (0.014% of wet weight). Magnesium in anterior, middle, and posterior thirds was 0.013, 0.029, and 0.033 mg, respectively; and whole worms contained 0.075 mg, or 0.061%

1 l0

GOODCHILD~

DENNIS~

xz;

AND

MOORE

o

d

d

A

A

A o

S

x

~ I:S

+l

88

~8 8

s

d

d

d

V

A

V

ol

+i

+1

~

"~ o

9

d A 4-1

o

d

d

V +1

,.~

V N '-d

4-1

N

,e..1 m c, o

N '6

s

d

d

u~ & o

A

V

A ~

+l ~1 8o8 o+l. o ,

~o+l.S q

g

~q+lo8.

g

s

N

~a

s

8

8,

E ,-~

o

d

d

d

A

V

V

~

8

FLAME PHOTOMETRIC STUDIES OF HELMINTHS (0.012% of wet weight). Total K in the thirds was 0.190, 0.446, and 0.390 mg, and in whole worms equalled 1.027 rag, or 0.836.,% (0.160% of wet weight). Total Na was 0.056, 0.164, and 0.177 rag, and in whole worms was 0.397 mg, or 0.323% (0.062% of wet weight). The composite sum of the four cations in whole freeze-dried H. diminuta was 1.591 rag, or 1.296% (0.247~ of wet weight). .12 .11

.08

.10

.07

.09 .08

L,E

0

.07 .06

.05

A

,05

I % Mq

%Ca

FIG. 1. Percent of calcium and magnesium in freeze-dried, 28-day-old adult Hymenolepis dirninuta. Mean values designated by mid.lines in diagonally barred areas which represent ~ 2 S.E.~.

111

DISCUSSION

Prior to the application of emission spectrophotometry to Ca determinations in Hymenolepis diminuta, three other methods were attempted in this laboratory without achieving consistent results; however, these methods proved reliable with materials containing Ca in adequate amounts (e.g., standards containing Ca, tap water, and Lab-Trol, a known standard marketed by a commercial supplier) .5 The first method, titration with the disodium salt of ethylenedinitrilotetraacetic acid (EDTA), following the procedure in the American Public Health Association, Inc. (1955) publication, gave with tapeworm samples (10 to 50 rag) such subtle endpoints with ammonium purpurate indicator that results were not reproducible. The second method, an E D T A titration following the procedure of Diehl and Ellingboe (1956) and using the Beckman/Spinco Model 150 Ultramicro Analytical System, also gave unreliable endpoints with calcein indicator. The third technique, the Ferro and Ham (1957a, b) colorimetric method, used chloranilic acid and EDTA. Beer's law is said to be applicable over a range from 4 to 50 mg % Ca, but to 5 Dade Reagents, Inc., Miami, Florida. 1.50_

1.00

_

.40_

.:50_

.9O

1.40 _

~Z

1.30 -

~z ~Z fJ,

r.

rJ

.80.

.20_

.70

.10 %K

1.20 -

A M P % Na

Uol

lAMP % Ca+ %Mg + % K + % No

FIG. 2. Percent of potassium and sodium and sum of percentages of four cations in freeze-dried, 28day-old adult Hymenolepis diminuta. Mean values designated by mid-lines in diagonally barred areas which represent • 2 S.E.~t.

112

GOODCHILD, DENIqlS~ AND MOORE

obtain even the lowest of these concentrations ash from 25 to 30 mg of dried tapeworms had to be transferred quantitatively in 0.5 ml of solvent. This was technically difficult, results were erratic, and for routine analyses even smaller samples of worms were desired. After resorting to flame photometry, CClaCOOH extraction and digestion with HNO3 and HC104 (5:1) followed by the standard addition and dilution methods of correction, as outlined by Dean (1960), were used with freeze-dried rat liver (which was available in abundance) to develop a reliable technique for determining Ca in tissues. However, interferences were encountered and results were inconsistent; e.g., three samples of liver gave the following percentages of Ca, 0.102, 0.026, and 0.013. Next Leyton's (1954) method of adding KH2PO4 to eliminate phosphate interference on Ca emission was tried with acceptable results, and was, accordingly, adopted as the method of choice. The amount of KH2PO4 added was such that the calculated concentration of P was at least one-half that of the Ca in the samples. In P : C a ratios greater than this there is no additional phosphate interference. Analyses of Ca in four samples of liver, and in five samples of tapeworm (from alloxan-injected hosts; results should not be compared with worms from normal hosts) gave percentage results as follows: 0.007, 0.006, 0.008, and 0.008 (liver); and 0.030, 0.024, 0.028, 0.028, and 0.027 (tapeworms). Twelve samples of CaCO~ and Ca~(PO4)2, individually and in mixtures of varying ratios, digested and analyzed for Ca gave 89% recovery. This represents a factor for Ca present divided by Ca found of 1.12 by which the amounts of Ca found in tapeworm samples were multiplied. Addition of varying amounts of the SO4 ion to Ca standards failed to affect emission at SO4:Ca ratios of up to 25:1. After the technique for determining Ca was perfected, attention was next directed to the determination of Mg, K, and Na. Dried liver and tapeworms were digested, diluted, and read against appropriate standards with consistency of results within acceptable limits of experimental error. Six samples each of

MgCO3, KC1, and NaC1, treated as were the samples, gave average percent recoveries of the elements as follows: 88 for Mg, 98 for K, and 91 for Na. These results gave factors of 1.14, 1.02, and 1.10 by which Mg, K, and Na, respectively, found in normal tapeworms were multiplied. Although phosphate is said not to interfere with Mg (Dean, 1960), it was considered expedient to test for such interference at the concentrations expected in our samples. Varying amounts of KH2PO~ were added to Mg standards in PO4:Mg ratios up to 10:1, but no interference was noted. "The sodium line at 589 m~t encounters very little spectral interference from other elements" (Dean, 1960); therefore, interferences from other cations or from anions were not studied in this work. Rubidium and lanthanum interfere with the emission of K at 769 mbt; however, these elements were not considered likely to be present in tapeworms, and, accordingly, no compensatory studies were made with these interferers. Calcareous corpuscles are conspicuous in some tapeworms (von Brand, 1952; von Brand et al., 1960) and constitute a sizeable percent (2.6 to 4.1) of the wet weight; moreover, analysis of dried, non-incinerated corpuscles in two species of adult worms (Hydatigera taeniae]ormis and Moniezia expansa) yielded 20.0 and 25.8~o Ca, and 10.5 ai:d 10.3% Mg, respectively. In H. taeniaeformis Ca constituted 0.52% and Mg 0 . 2 7 ~ of the wet weight. This species, thus, has 37.1 times as much Ca and 22.8 times as much Mg as are found in an equivalent weight of Hymenolepis diminuta. Comparisons of figures for Hydatigera taeniaeformis (von Brand et al., 1960) with fresh preparations of H. diminuta reveal a morphological basis for this difference. In the former, calcareous corpuscles are abundant and large; in H. diminuta they are sparse and small. The significance of these differences is unknown; however, if a chief role of the " . . . corpuscles is to buffer anaerobically produced acids" (von Brand et al., 1960), rats--which lack gall bladders--may utilize the continually flowing bile as a significant buffering system. The literature on known and suspected roles of inorganic materials in animal tissues

FLAME PHOTOMETRIC STUDIES OF HELMINTHS

is voluminous and will not be considered here. H e i l b r u n n (1952) has reviewed the literature for Mg, K, Na, and especially for Ca. I t was noted in the present work that Ca decreases significantly along an anteroposterior gradient; if, as proposed b y Bailey (1942), Ca is important in muscular contraction this decrease may be preparatory to loss of this function in gravid proglottids prior to their being sloughed from the strobila. D e t a c h m e n t of gravid proglottids may be further assisted b y decreases in Ca posteriorly since its absence affects the integrity of intercellular cement substances (Heilb r u n n , 1952). An interesting finding was the excess of K over Na, Mg, or Ca in dried tapeworms; K exceeded N a by a factor of 2.6, Mg by 13.7, and Ca by I1.I. Somewhat similar ratios occurred in red blood cells where K is 5.1 times as a b u n d a n t as Na, 18.6 times that of Mg, and 95.0 times as plentiful as Ca (Spector, 1956). I n serum, on the other hand, the q u a n t i t y of K is 0.03 that of Na, 2.33 that of Mg, a n d 0.81 that of Ca (Spector, 1956). Ratios of K : N a and K : M g are similar for dried tapeworms, rat liver, and red blood cells, b u t in tapeworms the a m o u n t of Ca is greater than in liver or red blood cells hence the K : C a ratio is much lower than in these tissues. Most organisms, at least those with internal fluids, contain total N a in excess over other cations. Formulae for isotonic physiological salines are a reflection of this disproportion. A partial explanation of this cationic difference in cestodes m a y be their lack of a conspicuous milieu intdrieur. F l u r y ' s (1912) determination of cations in Ascaris, which possesses a fluid-filled pseudocoelom, showed N a in excess over the other cations. REFERENCES AMERICAN PUBLIC HEALTH ASSOCIATION, INC. 1955.

Standard Methods for the Examination of Water, Sewage, and Industrial Wastes. American Public Health Association, Inc., New York. BAILEY, I. 1942. Myosin and adenosinetriphosphatase. Biochemical Journal 36, 121-139. YON BRAND, T. 1939. Chemical and morphological observations upon the composition of Macracanthorhynchus hirudinaceus (Acanthocephala). Journal o] Parasitology 25, 329-342.

113

VON BRAND, T. 1952. Chemical Physiology of Endo-

parasitic Animals. Academic Press, New York. YONBRAND,T. 1957. Recent trends in parasite physiology. Experimental Parasitology 6, 233-244. VON BRAND, T., MERCADO, T. I., NYLEN, M. U., AND SCOTT, D. B. 1960. Observations on function, composition, and structure of cestode calcareous corpuscles. Experimental Parasitology 9, 205-214. VON BRAND, T., AND SAURWEIN, J. 1942. Further studies upon the chemistry of Macracanthorhynchus hirudinaceus. Journal o] Parasitology 28, 315-318. DEAN, J. A. 1960. Flame Photometry. McGraw-Hill Book Company, Inc., New York. DIEHL, H., ANDELLINOBOE,J. L. 1956. Indicator for titration of calcium in presence of magnesium using disodium dihydrogen ethylenediamine tetraacetate. Analytical Chemistry 28, 882-884. DINNIN, J. I. 1960. Releasing effects in flame photometry determination of calcium. Analytical Chemistry 32, 1475-1480. FERRO, P. V., AND HAM, A. B. 1957a. A simple spectrophotometric method for the determination of calcium. American Journal o] Clinical Pathology 28, 208-217. FERRO, P. V., ANDHA~, A. B. 1957b. A simple spectrophotometric method for the determination of calcium. II. A semimiero method with reduced precipitation time. American Journal o] Clinical Pathology 28, 689-693. FLURY, F. 1912. Zur Chemie und Toxikologie der Ascariden. Archly ]iir Experimentelle Pathologic und Pharmakologie 67, 275-392. GOODCI-IILD,C. G., ANDKAGAN,I. G. 1961. Comparison of proteins in hydatid fluid and serum by means of electrophoresis. Journal o/Parasitology 47, 175-180. HEILBRUNN, L. V. 1952. An Outline of General Physiology. W. B. Saunders Co., Philadelphia. LEYTON, L. 1954. Phosphate interference in the flame photometric determination of calcium. Analyst 79, 497-500. SALISBURY, L. F., AND ANDERSON, R. J. 1939. Concerning the chemical composition of Cysticercus ]asciolaris. Journal o] Biological Chemistry 129, 505-517. SCHOFFER, W. H. 1932. Recherches physico-chimiques sur le milieu interieur de quelques parasites. Revue suisse de zoologie 39, 59-194. SPECTOR, W. S. (ed.). 1956. Handbook of Biological Data. W. B. Saunders Co., Philadelphia. WARDLE, R. A., AND MCLEOD, J. A. 1952. The Zoology of Tapeworms. University of Minnesota Press, Minneapolis.