17-Hydroxycorticosteroids and leucocytes in the blood of dairy cattle

J.

COMPo

PATH.

1957·

VOL. 67·

r6,'}

17-HYDROXYCORTICOSTEROIDS AND LEUCOCYTES IN THE BLOOD OF DAIRY CATTLE By

J. Y. F.

PATERSON

Department of Biochemistry, Unil'ersity of Licelpool INTRODUCTION

Shaw (1947), believing that bovine ketosis was similar in some respects to an adrenal cortical insufficiency, attempted the treatment of four cases of ketosis with an adrenal cortical extract and found a marked improvement in the condition of the animals. Subsequently Shaw, Hatziolos and Leffel (1950) found that cortisone was beneficial in the treatment of ketosis and observed that in ketotic cows the adrenal cortices and anterior pituitary showed evidence of response to stress. On the basis of such evidence they put forward the theory that bovine ketosis resulted from adrenal cortical insufficiency following stress reactions. Their evidence in support of this view is somewhat indirect, being concerned largely with "replacement therapy" (the treatment of ketosis with cortisone, corticotrophin and similar hormones) and the subnormal response, as judged by increase in blood glucose and decrease in circulating eosinophils, of ketotic cows to injections of corticotrophin or adrenaline (Shaw, Hatziolos, Leffel, Gill and Chung, 1952; Shaw, Brown, Gessert and Chung, 1954). Puntriano (1951) has put forward a similar theory of the aetiology of ketosis, and has found that the urinary excretion of glucocorticoids by ketotic cows is less than that of normal cows (Puntriano, 1952). The ketotic cow shows marked deviations from normality in blood composition, in particular showing marked hypoglycaemia and ketonaemia. If one accepts the findings of Shaw and of Puntriano, there should also be evidence of decreased adrenal cortical secretion of hormones, for example a decreased level of blood adrenal cortical Steroids. The present work was undertaken in the hope that observations on the sequence of these changes in blood composition might prove helpful in assessing certain of the current views on the aetiology of bovine ketosis. When it was begun, no reports had been published of estimations of adrenal steroids in the blood of dairy cows, but recently Robertson and Mixner (1954, 1956) have described the estimation of plasma 17-hydroxycorticosteroids in cattle. Since it was desirable that the observations should extend for some time before the appearance of clinical symptoms, it was decided to use as subjects cows with a history of ketosis in previous lactations, in the hope that some of these would develop ketosis in the lactation under study. Unfortunately, none of the animals studied developed ketosis, and so the findings reported here relate entirely to clinically normal

166

17-HYDROXYCORTICOSTEROlDS AND LEUCOCYTES IN BOVINE BLOOD

animals. The present paper concerns observations on leucocytes and on plasma I7-hydroxycorticosteroids: observations on blood glucose, ketones and volatile fatty acids will be given in a subsequent paper. METHODS

Estimation oj Plasma 17-Hydroxycorticosteroids

The method used was that of Bayliss and Steinbeck (1953) with certain modifications. It was found that emulsification during extraction of the plasma with chloroform could be avoided if the volume . of chloroform used for extraction was made at least twice as great as the volume of plasma. With this greater proportion of chloroform, the extraction of I7-hydroxycorticosterone added to plasma was complete with two extractions. It was found that unless chloroform extracts of bovine plasma were washed carefully with dilute sodium hydroxide the final methanol-sulphuric acid mixtures were turbid and quite unsuited to spectrophotometry. In the chromatography of plasma extracts a mixture of magnesium trisilicate. and 'Celite' was used as adsorbent, as described by Nelson and Samuels (1952). Procedure. Oxalated plasma, 60 mi., was extracted twice with chloroform, 150 mi., the mixtures being shaken for I min. in each extraction. The combined chloroform extracts were washed once with 0'1 N sodium hydroxide, 20 mi., and twice with water, 20 mi., each washing being back extracted with an equal volume of chloroform. The washed extract was dried over anhydrous sodium sulphate, filtered and evaporated almost to dryness at 40 to 45 °C. in vacuo. Magnesium silicate- Celite mixture (1:1 w/w), 1'5 g., was suspended in chloroform in a chromatogram tube fitted with a sintered glass disc (1 cm. dia., porosity 3) and the column packed under pressure, 5 lb. N 2/Sq. in. The plasma extract was applied to the column in chloroform, 5 mi., washed on with two 2 mi. portions of chloroform, and the chromatogram developed with chloroform, 25 mi., 2 per cent (v/v) ethanol in chloroform, 25 mi., and 20 per cent (v/v) ethanol in chloroform, 25 mi. The third eluate was collected and evaporated to dryness at 40 to 45 °C. in vacuo. The residue from this eluate was dissolved in methanol, 2'5 mi., and two 1'0 mi. portions taken. To one was added 62 per cent (v/v) sulphuric acid, 2'0 ml., and to the other 0'16 per cent (w /v) phenylhydrazine hydrochloride in 62 per cent (v/v) sulphuric acid. Reagent blanks and chromatogram blanks (from a column with no plasma extract added) were also prepared. The mixtures were heated at 60°C. for 30 min., and after cooling the optical densities were read at 410 miJ. in I cm. cells in a Unicam spectrophotometer. The calculation of results was as described by Bayliss and Steinbeck (1953). Using 17-hydroxycorticosterone as standard, the regression equation of the colour reaction was found to be: weight of 17-hydroxycorticosterone (iJ.g.) =0'06 + 58'9 X optical density. In twelve experiments the recovery of 17-hydroxycorticosterone added to plasma ranged from 73 per cent to 90 per cent, the mean recovery being 78'5 per cent. Leucocyte determination. Total leucocytes were determined as described by Boddie (1950) and differential counts were made as described by Coffin (1953)'

J.

Y. F. PATERSON

Collection of blood samples. Blood was collected from the jugular vein (1 3 b.w.g. needle) directly into wide mouth bottles containing ammonium oxalate-potassium oxalate mixture (3:2 w/w). All blood samples were collected at the same time of day, prior to afternoon milking, and all analyses were begun within about 45 min. of the collection of the samples. RESULTS

The animals studied were Ayrshire cows on farms near the University of Liverpool Veterinary Field Station. Six cows with a history of ketosis in their 1954/1955 lactations were selected, all being due to calve between November 1955 and April 1956. Sixteen cows with no previous history of ketosis were also available, ten of these being due to calve in the same period as the cows with a history of ketosis. These normal animals were selected so that each cow with previous ketosis was studied with two or three normal cows on the same farm, all being due to calve within a few days of each other. The animals were bled at approximately weekly intervals from ten weeks before the expected date of calving to ten weeks after calving, or for part of that period. Of the six cows with a history of ketosis, one aborted twin foetuses one month before the expected date of calving, and the results from this cow have been excluded from the consideration of results. None of the remaining five cows with a history of ketosis developed ketosis in the lactation under study. Since all the animals studied were clinically normal and healthy it seemed unlikely that any differences would be found between normal cows and cows with a history of ketosis. Plasma 17-hydroxycorticosteroids Comparison of normal cows and cows with previous ketosis. The levels of plasma 17-hydroxycorticosteroids for 16 normal cows (223 r esults) and 5 cows with a previous history of ketosis (81 results) are shown in Fig. I, from which it may be seen that most of the results are below 5 fLg. 17-hydroxycorticosterone per 100 ml. plasma. The mean and standard deviation for normal cows prepartum were 2·2 ± 1'4 fLg. 17-hydroxycorticosterone/100 ml. plasma, and for cows with previous ketosis 1'9 ± 1·0 fLg. per cent. For the difference between these means 't' was 1.069 for 124 degrees of freedom, P ca. 0'3. The mean and standard deviation for normal cows postpartum were 1'4 ± 0·8 fLg. I 7-hydroxycorticosterone/ 100 ml. plasma, and for cows with previous ketosis 1·2 ± 0'5 fLg. percent. F or the difference between these means 't' was 2 '067 for 176 degrees of freedom, P >0·2. It is, therefore, apparent that no marked difference exists between the two groups of animals. . The results from normal animals showed an apparent seasonal variation, and on analysis this variation was found to be significant. Some of the variation seen from month to month was undoubtedly due to fluctuations in the numbers of calvings per month. When the

168

17-HYDROXYCORTICOSTEROIDS AND LEUCOCYTES IN BOVINE BLOOD

results are arranged so that the frequency of calvings is more uniform the seasonal variation is still found (Table I). In the case of both prepartum and postpartum results, the values found in the summer months are significantly higher than those found in the winter months. FIGURE

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Plasma levels of 17-hydroxycorticosteroids (expressed as /-,g. 17-hydroxycorticosterone/ lOo ml. plasma) of cows with previous ketosis (. ) and of normal cows (0) before and after parturition.

For normal animals there is a highly significant difference between the postpartum results for the period July to September and those for the period October to December, 't' being 4.06 for 71 degrees of freedom, P
J.

169

Y. F. PATERSON

variation in the numbers of calvings is a contributory factor, particularly in the case of the prepartum results. When the results from normal animals obtained during the same periods as the results from animals with previous ketosis are compared, no significant difference is found (Table I). For subsequent consideration it will be assumed that no difference exists between the results from normal animals and the results from animals with previous ketosis. TABLE SEASONAL VAIUATlOl\ IN

PLAS~1A

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17-HYDKOXYCORTICOSTEROIDS

Comparison of normal cows and cows with previous ketosis. (Results expressed as "g. 17-hydroxycorticosterone/IOo m!. plasma). Prepartum results Period

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Changes in plasma I7-hydroxycorticosteroi ds bifore and qf'ter parturition. The mean values and standard deviations of plasma 17-hydroxycorticosteroids for all animals for 10 day periods over the period studied are shown in Table 2. There is an obvious tendency for the level of plasma 17-hydroxycorticosteroids to increase over the prepartum period, although the increase is most marked in the 10 days immediately prior to parturition. The mean for this period is significantly higher than the mean for the preceding 10 day period. There is a marked decrease in plasma 17-hydroxycorticosteroids at parturition, and thereafter there is a gradual decline until about 30 days postpartum, after which no further change

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occurs. These tendencies in the results are seen equally clearly in Fig. I. Considerable variation was seen however in the changes in plasma 17-hydroxycorticosteroids of individual animals as illustrated in examples A to E (Fig. 2). While some cows showed increasing plasma I7-hydroxycorticosteroids as pregnancy progressed, both the rate and extent were variable. In some the increase was reasonably progressive, as in B, whereas in others the greatest increase was seen immediately prior to parturition, as in A where there was a twofold increase in the 24 hr. period prior to parturition. About two-thirds of the animals showed increasing plasma 17hydroxy corticosteroids prepartum, while most of the remainder showed more or less uniformly high levels over the prepartum period studied, as in C. Four cows showed a decrease in plasma 17-hydroxycorticosteroids prepartum, as in D and E. In these cases the decreases occurred within two weeks of parturition, and followed a general increase. FIGURE 2 4

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172

17-HYDROXYCORTICOSTEROlDS AND LEUCOCYTES IN BOVINE BLOOD

In almost all cases there was a sharp decrease postpartum, the decrease being usually very rapid. In those cases bled within 12 hours before parturition and within 12 hours after parturition, most showed a marked decrease in the 24 hour period. After the initial postpartum decrease. some cows showed reasonably steady levels as in B or generally decreasing levels, as in C. About two thirds of the animals showed such postpartum changes; the remaining third, however, showed a subsequent temporary increase postpartum. These peaks occurred in the period 2 to 4 weeks postpartum. While about two-thirds of the individual animals studied showed prepartum and postpartum changes essentially similar to the mean picture, some cows showed deviations, notably decreases prepartum and peaks postpartum.

Changes in Leucorytes bifore and after Parturition For most of the blood samples in which plasma I7-hydroxycortico steroids were determined, total and differential leucocyte counts were also estimated. About three-quarters of the animals showed prepartum increases in total leucocytes, the greatest being observed in the two weeks before parturition. The remaining animals showed no marked change in totalleucocytes before parturition, but as none of these was observed at less than five days prepartum, they too may have changed in the immediate prepartum period. The marked increase seen at about 10 days prepartum is significant, 't' = I ·82 for 41 degrees of freedom, P >0·05. In about 20 per cent of cases the prepartum increase in total leucocytes extended to a day or two after parturition, but all showed a marked decrease in the immediate postpartum period, which was significant, 't' = 2' 19 for 52 degrees of freedom, P >0'02. While the mean postpartum decrease is followed by an increase to the very high prepartum level, and by a subsequent decrease to a relatively constant level, there was some variation shown by individual animals. In some, the postpartum decrease was so profound and of sufficient duration as to constitute a very marked depression in total leucocytes, whilst in others the decrease was not very marked. Similarly in the subsequent increase, some animals showed such a marked increase that there was a well defined peak prior to the attaining of a constant level, whilst in others there was no marked peak. The mean values, given in Table 3, are a reasonably good representation of the levels of most individual animals, the notable variations being the continuance of the prepartum increase for a short time postpartum, and the extent of the postpartum decrease and subsequent increase. Changes in neutrophils were in essential very siInilar to the fluctuations in total leucocytes, and in most animals the more prominent changes in total leucocytes could be accounted for by the neutrophils, although in the postpartum decrease in total leucocytes a transient lymphopenia was contributory in some cases.

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±S.D.

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LEVELS OF LEUCOCYTES BEFORE AND AFTER PARTURITION

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174

I7-HYDROXYCORTICOSTEROIDS A N D LEU COCYTES I N BOVINE BLOOD

Most animals showed a t endency to increasing neutrophils prepartum, although in some cases the change was noticeable only in the two weeks before parturition, and in some the increase extended to a day or two after parturition. In all cases there was a marked decrease postpartum, and as in total leucocytes, some animals showed a very profound decrease before attaining a constant postpartum level, while others showed a marked peak after the postpartum decrease. For the difference between the means for 20-11 days and 10-0 d ays prepartum 't'=3'05 for 41 degrees of freedom, P > o·OOI. For the decrease at parturition 't' =1'74 for 52 degrees offreedom, P > 0·05. The changes at 10 days and 20 days postpartum are also significa nt at 5 per cent and I p er c ent respectively. There were no m arked changes in lymphocytes. Most animals showed a very slight lymphopenia in the immediate prepartum period, although in some cases the lymphopenia was more noticeable in the immediate postpartum period. Most animals showed no marked change in eosinophils until the immediate prepartum period, when there was an eosinopenia. In some the decrease in eosinophils continued into the postpartum period, and in some the greatest decrease in eosinophils was seen in the period immediately after parturition. While for the mean the decrease prepartum is significant, the most significant change is the decrease at parturition; 't' = 2'75 for 52 degrees of freedom, P > o·OOI. The correla tion between plasma q-hydroxycorticosteroids and eosinophils was calculated for each animal, and of the 21 values of correlation coefficient so obtained 14 were negative, ranging from r=-0'104 to r = -o'730, while the remaining seven values were positive, ranging from r=+0.II3 to r = +0·379. A pooled estimate of the correlation coefficient was obtained using Fisher's z transformation and was found to be r =-0'291. Thus only in a few cases was there good correlation between plasma 17-hydroxycorticosteroids and eosinophil levels, and from the pooled estimate correlation was rather poor. DISCUSSION

From this work it would appear that the level of 17-hydroxycorticosteroids in the peripheral blood of cattle is quite low, and considerably lower than the level in man. Bayliss and Steinbeck (1953) found a range of 5.0 to 20'4 fLg. per cent for normal nonpregnant women, and Bayliss, Browne, Round and Steinbeck (1955) found values of lItO 42 fLg. per cent for the last three months of pregnancy in women, and values of 3 to 42 fLg. per cent for the early postpartum period. In the present work the values for the last 2 to 3 months of pregnancy in cows ranged from 0'3 to 9.1 fLg. per cent, and postpartum from 0' 3 to 4'5 fLg. per cent. These values are lower than the values found by Robertson and Mixner (1956) who

J.

Y. F. PATERSON

175

report values of 2'14 to 8'40 postpartum in cows, and valucs of 6'90 to I7·62 for the last six weeks of pregnancy. Forsius and Haikonen (I955), using the method of Bayliss and Steinbeck (1953) found values of 0 to 4' 5 fLg. per cent in sheep. Robertson and Mixner (1956) attempted to apply to bovine plasma the method of Nelson and Samuels (I 952) but found that the measurement of absorption at 4IO mfL of the steroid phenylhydrazone was impaired by excessive chromogenesis by the sulphuric acid of the reagent. To avoid this they adopted ethyl acetate in place of chloroform for the extraction of plasma, and omitted the chromatography of the plasma extract. While in the present work it was found that the application to bovine plasma of the method of Bayliss and Steinbeck (1953), which is an improved modification of the method of Nelson and Samuels (I952), gave final reagent mixtures unsuited to spectrophotometry, this was believed to be due to turbidity which could be avoided by washing the plasma extract with dilute sodium hydroxide. No comparison has yet been made of the present method with that of Robertson and Mixner (1956) and therefore no reason can be given for the discrepancy in results. A considerable number of steroids are present in adrenal cortical tissue, but it now seems clear that only a few are found in adrenal venous blood, and so presumably in peripheral blood. The adrenal cortical steroids of biological importance have certain structural features in common, namely an a ketal side chain and a 6 4 3ketone. Hechter, Zaffaroni, Jacobsen, Levy, Jeanloz, Schenker and Pincus (195 I) isolated a number of a ketolic steroids from blood perfused through bovine adrenals. Corticosterone and 17-hydroxycorticosterone were present in considerable amounts, and cortisone and I I-dehydrocorticosterone were present in smaller quantities, as were a number of unidentified substances, possibly reduction products of these other steroids. Bush (1953) in a study of adrenal venous blood, found in all the species studied that corticosterone and/or 17-hydroxycorticosterone constituted some 85 to 100 per cent of the 6 4 3-keto steroid detected. In bovine adrenal venous blood corticosterone and I7-hydroxycorticosterone were present in approximately equal amounts. It would therefore seem probable that in bovine peripheral blood the main biologically active component is I7-hydroxycorticosterone, and consequently estimation of blood I7-hydroxycorticosteroids should provide a reliable index of adrenal cortical function. Blood I7-hydroxycorticosteroids can be estimated by the methods dealt with at the beginning of this discussion. It seems probable that the I7-hydroxycorticosteroids of blood are present in the plasma only (Morris, I953), and these methods use plasma. The methods have in common their colour reaction (Porter and Silber, I950), which is based on the reaction between the steroid and phenylhydrazine in strong sulphuric acid, and which is claimed to

17 6

17-HYDROXYCORTICOSTEROIDS AND LEUCOCYTES IN BOVINE BLOOD

be specific for a ketolic steroids with a hydroxyl group at C 17. From the work of Bush (1953) the only such steroid present in bovine blood in appreciable amount should be I7-hydroxycorticosterone, although there may also be present metabolic products of 17hydroxycorticosterone. Bayliss and Steinbeck (1953) in an attempt to verify the specificity of their method, found that about half of the 17-hydroxycorticosteroid estimated was 17-hydroxycorticosterone, the remainder possibly being reduction products of 17-hydroxycorticosterone or of cortisone. If similar substances are present also in bovine peripheral blood, their inclusion in the material estimated does not detract from the value or specificity of the method. The results presented in this paper show clearly an increased adrenal cortical activity in pregnancy in dairy cows. It is difficult to choose from these results a value of plasma I7-hydroxycorticosteroid for a "normal cow", but if the relatively constant value of about 1·0 fLg. per cent at 5 to 10 weeks postpartum is taken as normal, then increased levels were observed throughout the last ten or eleve.ll weeks of pregnancy. This work has not determined at what stage of pregnancy the increase in plasma I7-hydroxycorticosteroid begins. Bayliss, Browne, Round and Steinbeck (1955) found that in women the increase in adrenal cortical activity began at about the third month of pregnancy, and also found some variation in the changes shown by individuals. Somewhat similar variation was noted among individual cows in the present work. Although most cows showed a decrease in adrenal cortical activity at about parturition, too few were examined less than 24 hours prepartum and immediately postpartum to decide how closely the decrease in plasma 17-hydroxycorticosteroids was associated with parturition. The changes in the first few weeks after parturition are of interest, particularly the peak levels of plasma 17-hydroxycorticosteroid shown 2 to 3 weeks postpartum by about one third of the cows. This peak appeared to be associated with maximum lactation, but it does not necessarily follow that it indicates a response to a 'lactation stress'. In fact the most marked increase in steroid was observed in the cow with the lowest milk yield (about 25 lb. per day) and with almost constant milk yield for the first five weeks postpartum, while cows giving milk yields of about 60 lb. per day and showing a very marked peak in milk yield showed little or no increase in plasma I7-hydroxycorticosteroid. The finding of an apparent seasonal variation in plasma 17hydroxycorticosteroid was made too late for any attempt to be made to determine the reason for the variation. That the highest levels were found in the summer months, and that quite a sharp change was seen about October might suggest temperature as a contributory factor; particularly since the summer of 1955 was on the whole rather warm and dry. An alternative explanation might be that in summer months the animals were more active, and in fact were bled

J.

Y. F. PATERSON

soon after they had walked from the pasture to the dairy. In the winter months they were of course indoors and thus were not exercised just before bleeding. The changes in leucocytes at parturition in the normal animal are well known, and it has been shown that similar changes in leucocytes may be elicited in the non-parturient animal by the administration of corticotrophin (Smith and Niedermeier, 1953; Merrill and Smith, 1954). It was, therefore, to be expected in the present work that where increases in plasma 17-hydroxycorticosteroids were observed these would be associated with changes in leucocytes. The association observed in individual animals varied considerably, an obvious example of this being the positive correlation between plasma 17-hydroxycorticosteroids and eosinophils shown by 7 cows. When it is borne in mind that the bleedings were done at weekly intervals it is possibly not surprising that some variation was found, and it might be expected that analysis at more frequent intervals would show better correlation between plasma 17-hydroxycorticosteroids and leucocytes. From the results given in this paper it would appear that any attempt at assessing adrenal function on the basis of say one or two counts of eosinophils might be subject to considerable error. There is such a widc variation in individuals in leucocyte levels that there could be no question of assessing adrenal cortical function other than by frequent counts over a period of time. Changes then observed in leucocytes might form a reasonably reliable qualitative index of change in adrenal cortical function, but such observations would have no quantitative significance. The use of the eosinopenic response to adrenaline or corticotrophin is becoming commonly used as a means of assessing adrenal cortical function. Shaw et al. (1952), used such tests in ketotic cows and found that in the ketotic cow the adrenal cortical response to adrenaline and to corticotrophin was subnormal. No attempt has yet been made to determine the reliability of such tests in dairy cows, but Hunter, Bayliss and Steinbeck (1955) found in man that while adrenaline invariably produced a marked eosinopenia, it was difficult to relate such changes to changes in plasma I7-hydroxycorticosteroids estimated simultaneously. The only estimations of plasma 17-hydroxycorticosteroids in ketotic cows so far published were by Robertson, Lennon and Mixner (1955) who found that in ketotic cows the plasma 17hydroxycorticosteroids were slightly higher than normal. This finding is not in accord with Shaw's concept that ketosis results from adrenal cortical failure. It is intended to pursue the original objective of this present work, which is to determine how adrenal cortical function in the ketotic cow differs from normal, and to determine the sequence of changes in adrenal cortical and other metabolic functions.

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17-HYDROXYCORTICOSTEROIDS AND LEUCOCYTES IN BOVINE BLOOD CONCLUSIONS

Plasma I 7-hydroxycorticosteroids were determined and leucocyte counts were made in 22 dairy cows for some time before and after parturition. The mean values of plasma 17-hydroxycorticosteroids were higher prepartum than postpartum. The highest values were found I to 2 weeks prepartum and there were marked decreases at parturition. Total leucocytes and neutrophils showed significant increases I to 2 weeks prepartum, with sharp decreases at about parturition. There was a significant eosinopenia I to 2 weeks prepartum. Changes in lymphocytes were not significant. ACKNOWLEDGMENTS

The expenses of this work were in part defrayed by a grant from the Agricultural Research Council. The author is grateful to Prof. R. A. Morton, F.R.S. for his interest in this work; to Prof. J. G. Wright and to Prof. E. G. White for laboratory accommodation and facilities at the University of Liverpool Veterinary Field Station; to the clinical staff of the Field Station for their co-operation; and to Miss M. Ducker for leucocyte determinations. The author is deeply grateful to the managers and dairy staffs of Ashfield Hall Farm, Dunstan's Farm, Hanns Hall Farm, and Puddington Home Farm for their willing and unfailing cooperation. REFERENCES

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Shaw,]. C., Hatziolos, B. C., and Leffel, E. C. (I950). J. Amer. vet. med. Ass., 87, 73. Shaw,]. C. Hatziolos, B. C., Leffel, E. C., Gill, W. M., and Chung, A. C. (I952). J. dairy Sci., 35,497. Shaw, ]. C., Brown, R. E., Gessert, R. A., and Chung, A. C. (I954). Ibid., 37, 66!. Smith, V. R., and Niedermeier, R. P. (I953). Ibid., 36,597. [Receivedfor publication, August I6th, I956]