- Email: [email protected]
Some observations on glucose, ketone bodies and volatile fatty acids in the blood of dairy cattle
J.
COMPo
PATH.
1957. VOL. 67.
33 1
SOME OBSERVATIONS ON GLUCOSE, KETONE BODIES .AND VOLATILE FATTY ACIDS IN THE BLOOD OF DAIRY CATTLE By
D. HORROCKS and ]. Y. F. PATERSON Department qf Biochemistry, University qf Liverpool
INTRODUCTION The determinations of blood glucose, ketone bodies and volatile 'fatty acids described in this paper were carried out simultaneously with the estimations of plasma 17-hydroxycortico-steroids described by Paterson (1957). It was intended to extend observations by ,earlier workers on changes in blood composition at parturition and in early lactation in normal cows.· It has been reported that the blood level of glucose increases just before parturition, decreases immediately after parturition and thereafter is constant for one to four weeks (Godden and Allcroft, 1932; van Soest and Blosser, 1954). Knodt, Shaw and White (1942) and Sampson and Boley (1945) have reported that the blood level of ketone bodies shows no change at parturition, but is higher in early than in late lactation . .No observations on blood levels of volatile fatty acids in pregnancy and lactation appear to have been reported. Shaw (1943) studied the development of ketosis by estimation -of blood glucose and ketone bodies in early lactation in cows from ·a herd with a high incidence of ketosis. In cows developing ketosis the decrease in blood glucose following parturition continued for four to ten days, during which time blood ketone bodies increased markedly, until clinical symptoms of ketosis appeared. It was intended to extend Shaw's work by observations on cows which had a history of ketosis in previous lactations and which might thus be expected to develop ketosis in the lactation under study. None of these animals developed ketosis in the period of study however and so this paper deals only with clinically normal animals. METHODS Blood glucose. Miller and van Slyke (1936). Plasma volatile fatty acids. Conway and Downey (1950). Blood ketone bodies. Blood ketone bodies were estimated by a modification of the method of Greenberg and Lester (1944). The reflux units described by these authors were found inconvenient in routine operation and instead the conversion of ~-hydroxybutyric acid and acetoacetic acid to acetone was carried out in glass-stoppered test tubes heated in a boiling water bath. The stoppers, lubricated by a silicone grease, were retained in the tubes by a rubber-cushioned metal plate. The time of ·extraction of acetone 2:4 dinitro-phenylhydrazone with carbon tetra·chloride was increased to 25 min. since IO min. was found to be in-
332
BIOCHEMICAL EXAMINATION OF THE BLOOD IN CATTLE
sufficient for complete reaction. The 2:4 dinitro-phenylhydrazine· reagent was washed with carbon tetrachloride to give lower blanks as· recommended by Pennington (1954). f3-hydroxybutyric acid was at first estimated by difference from determinations of total ketones and of acetone and acetoacetic acid, but this procedure was eventually found to be unreliable at normal blood levels of these substances due to the inconsistent recovery of acetone and acetoacetic acid in the method for total ketones. The method described by Greenberg and Lester ( 1944) for the direct determination of f3-hydroxybutyric acid was found to be reliable. Unfortunately most of the determinations of f3-hydroxybutyric acid were made by the former method, and therefore no results from estimations of f3-hydroxybutyric acid have been included in this paper. RESULTS
In this and following sections plasma volatile fatty acids will be referred to as plasma V.F.A. The cows in this study were those used by Paterson ( 1957) and since all remained clinically normal throughout the period of study it seemed unlikely that any differences would be found between the normal cows and those with a history of ketosis. in previous lactations. Comparison of Normal Cows and Cows with Previous Ketosis
Since seasonal variations occurred in the blood levels of glucose,. ketone bodies and V.F.A. in normal cows, the results from cows' TABLE
I
COMPARISON OF BLOOD LEVELS
Glucose, ketone bodies and volatile fatty acids in normal cows and cows with previous ketosis Prepartum November-April Cows with previous ketosis Normal cows
Glucose (mg,% blood) Mean ± S,E, (no. of results) Sig. of difference Plasma V.F.A, (mg. acetic acid %) Mean ± S.E. (no. of results) Sig. of difIe rence Acetone and Acetoacetate (mg. acetone % blood) Mean ± S.E. (no. of results) Sig. of difference
Postpartum January-May - - - -Cows with previous ketosis Normal cows
- - - - - - - - - - - - ---- - 43 '5±7'3 (3 7)
44'4±6'I (37)
't' = 0·64, P<0·6
------
7' 2 ±2'4 (38)
7'9±2'5 (40 )
't'= , '28, P <0'3
2'5 ± I'0 (38)
2'O±0'9 (39)
47,6±6'4 4 8 'O±5'4 (45) 1_~9_)_ _ 't' =o '28, P
6'9±3 '0 (44)
6'6±3'7 (59)
' t' = 0 '38, P < o,8
2'4±1'3 (46)
- - - - - - - - - - - - - -- -- 't' = 1'94, P
- - -- -
2'2±"0 (61)
't' = 0'90, P<0'4
D. HORROCKS AND
J.
Y. F. PATERSON
333:
with previous ketosis are compared only with those from normal cows during the same months, November to May. From Table I it can be seen that no differences in the blood levels of glucose,. ketone bodies and V.F.A. were found between normal cows and cows with a history of ketosis in previous lactations. In the following considerations the results from cows with previous ketosis are therefore included with those from normal cows. Changes bifore and after Parturition The mean blood levels of glucose, ketone bodies and V.F.A. for 10 day intervals during the period of study are given in Table 2. The increase in blood glucose between 20 to I I days and 10 to 0 days prepartum is significant ('t'=2·18, P<0.05) as is the decrease between 0 to IO and II to 20 days postpartum ('t'=2·38, P<0.05). No other significant changes in blood glucose level were seen in the period of study. While plasma V.F.A. show a lower level at 0 to 10 days prepartum and a higher level at I I to 20 days postpartum, no significant difference was found between any two successive IO day periods. Postpartum, the mean level of V.F.A. for the period 0 to 30 days (7· I ±3· 3 mg. per cent) is significantly higher than the mean level for the period 31 to 70 days (5·9 ±3·0 mg. per cent), 't' =2·63, P< 0·01. No significant changes occurred at any time in the blood level of ketone bodies. Thus the overall picture is that the blood level of glucose increases just before parturition, and decreases thereafter; plasma V.F.A. levels are slightly higher in the first few weeks of lactation than subsequently, and ketone body levels show no change in the first 10 weeks of lactation. However, while most animals showed such a picture, there was some variation among individuals~ particularly in the postpartum period. The results for four individual animals (Fig. I) show the extent of this variability. Of the 2 I cows used in this study 14 showed an increase in blood glucose before calving followed by a decrease soon after calving (examples A, B and D, Fig. I). Four cows showed no apparent change in blood glucose over the period of parturition (example C). There was some variation in the extent of the decrease in blood glucose after calving and in the constancy of the postpartum glucose level. Of the 14 cows showing a decrease in blood glucose soon after calving, 8 showed a decrease to levels below 45 mg. per cent glucose, in some cases the decrease being very striking as in example A. Following these lower levels there was an increase to a relatively constant level. In others, after the decrease at parturition, the blood level of glucose was constant (example B) or increased slightly. Of the 7 cows which showed no appreciable change in blood glucose around parturition 6 showed a transient decrease to rather low levels 3 to 5weeks after calving (example C). Thus two-thirds of the individual
Mean ± S.E, (no, resu lts)
.
( 12)
2'O ±
I 'I
6'8 ± I'7 ( 12)
Plasma VFA (mg, acetate %) Mean ± S,E, (n o, results)
Acetone and acetoac etate (mg. acetone %)
46 '6±6 '3 ( II )
Glucose (mg, % blood) Mean ± S,E, (no, results)
70.6,
2' 1 ±o'7 ( 12)
6'6 ± I'5 ( 12)
45 '4 ± 4'5 (12)
60-51
TABLE 2
o' l± o'g ( 15)
7'O±2'3 ( 15)
44'2±4'7 (13)
2'g ± I ' 2 (2 1)
7'3 ± 3'2 (21)
42'8 ± 4 '8 (21)
----
.
2'7 ± I ' 1 ( Ig)
(20)
-
6'B±2'8
44'5±7' 7 ( 18)
2 '4 ± I 'o (21)
6'5±3'4 ( Ig)
46 'I±6'I (18)
20-II
2'7 ± 1'0 (27)
5'6±3'O (25)
52'4 ±8 '6 (27)
10-0
2 '7± "1 (3 2)
6'7 ± 2'6 (3 1)
5 I'7± 9 '8 (30)
0-10
3'2±2'6 (2g)
8'2±3'9 (27)
46 '1 ±5'g (2g)
11-20
2,6±I '3 (33)
6'6 ± 2 'g (32)
45'8±7'8 (32)
21-30
2'6± I 'O (23)
5'4±2'6 (24)
4 8 'I ± 5" (24)
4I -50
5,-60
2'5 ±I' 7 (23)
5 'g ±2'6 (23)
2'3± I 'O (2 1)
6'7±3'7 ( 17)
50'3 ± 16,8 48'1 ±4'2 (22) (2 1)
3 '-4 0
30 - 21
4 0-3'
50-4 l
D ays after parturition
D ays hefore parluriliQn
Clucose, ketone bodies and volatile fatty acids during pregnancy and lactation
CH ANGES I N THE BLOOD LEVELS
2'8± I' 7 (21)
5'7 ± 3'3 ( Ig)
48'4 ± 7'I (20)
6'-7°
~
C'l
Z
oI::)
o
t>:I t:D t'"
:J:
..,
o z o..,
~
~
t'"
C'l
I
t:D
o
~
t.>:>
(..>0
D. HORROCKS AND
J.
335
Y. F. PATERSON
cows showed a transient decrease in blood glucose to a moderately low or low level in the first ten weeks of lactation. Fig.
I.
FIGURE I EXAMPLES OF RESULTS FROM INDIVIDUAL COws 00
GLUCOSE
::E~
20
20V I
12
12
40
... 0-%
-
.V FA
KETONES
VFA
~VFA MG.%
"......... - ~ --~ . -------!-.. 50
GLUCOSE
MG%
[----+____ "__...
0 _____ 0 _0_ _ 0
30t:
I
,--./
70
so
C
D 12
KETONES &VJA.
_. . . . .1-.....___
... 0%
~
~
0
~
w
ro
~ m ~ ro ~ ~ 0 ~ w DAYS BEFORE OR AFTER PARTURITION
0 ' · - - -. •
ro
~
~
~
About half of the cows showed a decrease in the plasma level of V.F.A. before parturition. After calving, half of the cows showed transient high levels ofV.F.A. (examples Band C, Fig. r). Five of these showed a fairly well defined peak in V.F.A. (example B) but in six there was much fluctuation in the V.F.A. level (example C). Ten of these eleven cows showing high levels of V.F.A. postpartum showed also transient decreases in blood glucose; in six cases the lowest level of glucose was approximately coincident with the highest level of V.F.A., as in example C. Four cows showed a general increase in plasma V.F.A. throughout the postpartum period studied (example A) and four showed a general decrease (example D). Only six cows showed any appreciable change in the blood level of ketone bodies, and these showed transient high levels approximately coincident with a high level of plasma V.F.A. postpartum (example C). No pattern of agreement between changes in blood levels of these substances and changes in milk yield or the level of milk yield could be seen. Seasonal Variation The results showing seasonal VarIatIOn in the blood levels of glucose, ketone bodies and V.F.A. are given in Table 3. Both prepartum and postpartum the blood level of glucose was significantly higher in the period June to September than in other periods.
c
~
336
BIOCHEMICAL EXAMINATION OF THE BLOOD IN CATTLE
When considered by calendar months (Fig. 2), blood glucose is seen to be highest in July, to decrease progressively until December and then to increase slightly between January and March. Fig.
q
2.
-FA
---J(B
----
-
I--
r-
zc( ~~50 ~
III
840 3
"
30
1--1--
r-
- -
-
Jy Au sP· Oc No DE JA Fe MR AP My MONTH
FIG. 2 : SEASONAL VARIATION I N BLOOD COMPOSITION
The plasma level ofV.F.A. prepartum was significantly lower in the period June to September than in the period October to December, and was lower in this period than in January to April. From Table 2 it would appear that the plasma level ofV.F.A. postpartum was no different for the period July to September than for the period October to December. However among the cows studied in the period July to September was one which, although remaining clinically normal, showed very high levels of ketone bodies (14 mg. per cent) and V.F.A. (17 mg. per cent) for a short time. If the results from this animal are omitted from the results for the period July to September, the mean 'plasma level of V.F.A. is decreased to 4'9 ±2·3 mg. per cent and while this is not significantly different from the mean for the period October to December, the overall postpartum picture now resembles more closely the prepartum picture. From Fig. 2 it can be seen that plasma V.F.A. levels are highest in the period,December to March, i.e. approximately when blood glucose levels are lowest.
't'=O'2 7,
Sig, of difference
2'9±I'5 (4:»
I
6'4±2'7 (49)
[
2 'O±O '9 (53)
't'=2'52, P
7'6±2'S (53)
't'=o'59, P < o,6
46 'o±8,o (50)
't'=4'73, P
45'o± 10 '2 (48 )
P<~
't'=2,64, P < O'02
Sig, of difference
2'g±I'2 (20)
4'5±2'4 (23)
Plasma VFA (mg, acetic acid %) M ean ± S,E, (no, of results)
Acetone and Acetoacetate (mg, acetone % blood) Mean ± S,E, (no, of results)
't' = 2,6" P < O'02
5 "4± 8'o (23)
't'=2'27, P
3'g±2'5 (32)
't'=O'20, P
6'1±3'8 (3 0 )
I
I
3'O±O'7 (45)
I
5'9±2'4 (49)
I
4 6 '8±8'4 (51)
't'=3,6g, P
54'2±g'4 (30)
47'1±6'1 (92)
January-May
7'3±3 '2 (92)
I
't'=3 '40, P
2'2 ± I" (g6)
't'=2'79, P
I
't'=O'22, P
October-December
July-September
October-December
January-April
Postpartum
Prepartum
Glucose, ketone bodies and volatile fatty acids,
Sig, of difference
Glucose (mg, % blood) Mean ± S,E, (no, of results)
June-September
TABLE 3 SEASONAL VARIATION IN THE BLOOD LEVELS
-
-
!::l
-..J
c..J c..J
~
~ ~
!'l
:<
':-'
t1
Z
>
g;
oC'l
~
~
o
:I:
338
BIOCHEMICAL EXAMINATION OF THE BLOOD IN CATTLE
The prepartum blood level of ketone bodies was higher in the period June to December than in the period January to April (Table 3). The postpartum level was higher in July to September than in other periods. If the results from the cow showing rather higher than usual levels of ketone bodies and V.F.A., are excluded from the total results for this period, the mean for this period becomes 3.2 ±1·4 mg. per cent which is little different from the mean for the period October to December. From Fig. 2 it can be seen that blood ketone body levels are lowest in the period January to April. DISCUSSION
The determinations of plasma volatile fatty acids described in this paper represent an extension of observations by earlier workers on the composition of bovine blood in pregnancy and lactation. The method used gives good recoveries of formic, acetic and propionic acids which, according to Annison (1954), are the volatile fatty acids present in bovine plasma. It was not possible in the present work to confirm the identity of the acids estimated, but it is hoped to do so in subsequent work. Annison has found that of the total V.F.A. in bovine plasma 90 per cent is acetic acid and the values found by him (0·8 mM V.F.A. per litre=4·8 mg. per cent acetic acid) are not greatly different from the mean value in the present work (6.6 mg. per cent acetic acid) although in the present work the normal range was found to be quite large (I to 17 mg. per cent). The seasonal variation in the blood levels of glucose, ketone bodies and V.F.A. in normal cows is of interest. A seasonal variation in the blood level of ketone bodies has been reported by Sampson and Boley (1945) who found higher levels in June and July than in November to January. These workers observed their animals only during these two periods whereas in the present work the period of observation was continuous from June to May. Our finding that blood ketone body levels are lowest in the months January to April is therefore not in conflict with the finding of Sampson and Boley. In contrast Knodt et al. (1942) have reported that blood ketone body levels are highest in winter, but they considered that the increase was due primarily to winter feeding of silage to the animals studied. Eden and Green (1940) reported that blood ketone body levels in a herd of 60 cows were higher in March than in July, a finding contrary to that in the present work and in the work of Sampson and Boley (1945). It is unfortunate that the ketone body levels reported in the present paper comprise only acetone and acetoacetic acid, but it is considered unlikely that the omission of ,8-hydroxybutyric acid would account for the discrepancy with the findings of Eden and Green. No explanation can be offered for this variation in ketone body levels which is contrary to what might be expected from the seasonal
D. HORROCKS AND
J.
Y. F. PATERSON
339
variation in blood glucose levels. That the blood level of glucose is lowest in winter (December and January) is in accord with the seasonal incidence of ketosis in this country, most cases occurring in the winter months (Allcroft, 1947). While the aetiology of ketosis is in some respects still obscure it is clear that the development of a hypoglycaemic condition is one stage in the development of ketosis, and it is possible that the low winter levels of glucose observed in this work indicate a tendency to the development of hypoglycaemia. It is unlikely that the seasonal change in blood glucose is related to the seasonal change in plasma 17-hydroxycorticosteroids reported previously (Paterson, 1957), for while the maximum blood level of glucose coincides with the maximum level of steroid in June and July, there the apparent correlation ends. There may be a relationship between the lower blood glucose in winter and the higher level ofV.F.A. at that time, although there is nothing in the present work to show that one is the consequence of the other. An increase in blood glucose before calving, and a decrease after calving were first reported by Godden and AIlcroft (1932), and have subsequently been reported by Shaw (1943) and by van Soest and Blosser (1945). From the results given by Shaw the postpartum decrease in blood glucose can lead to transient lower levels of about 40 mg. per cent glucose, and from the results of van Soest and Blosser transient lower levels of 36 to 44 mg. per cent may be found soon after parturition. In the present work an increase in blood glucose level at parturition was seen in two thirds of the cows studied, and in the same proportion of animals there were seen also decreases postpartum to transient low levels of glucose, these lower levels ranging from 23 to 45 mg. per cent glucose. The high levels of blood glucose at parturition may be related to the increase in plasma I7-hydroxycorticosteroids at that time (Paterson, 1957) for in most cows changes in blood glucose were parallel to changes in corticosteroids. Boddie (1935) however, detected lactose in the urine of parturient cows and suggested that the increased level of blood sugar at calving might be due to resorption of lactose from the udder. No explanation can be offered for the transient low levels of glucose postpartum observed in the present work and by earlier workers. The rate and extent of the postpartum decrease in blood glucose in normal cows are, however, considerably less than in those cows developing ketosis observed by Shaw (1943). He found that the decrease in blood glucose just after parturition continued for 4 to IO days, the final levels being 15 to 20 mg. per cent glucose, but in the present work a similar decrease was seen in only one cow (example A, Fig. I) whose blood glucose level decreased from 57 mg. per cent to 29 mg. per cent in one week. In the present work only three cows showed transient higher ketone body levels (5 to 6 mg. per cent acetone) associated with lower blood glucose levels. Shaw found that during the development
340
BIOCHEMICAL EXAMINATION OF THE BLOOD IN CATTLE
of a marked hypoglycaemia blood ketone body levels increased rapidly until clinical symptoms ofketosis appeared. It would appear that in most normal cows ketone body levels undergo no appreciable change. The results presented in Table 2 and in Table 3 suggest that changes in blood glucose level might be reflected to some extent by changes in plasma V.F.A. level. In a few cases of ketosis where there was an opportunity for blood examination it was found that very high plasma levels of V.F.A. were associated with hypoglycaemia and ketonaemia (unpublished results). However while there might be some general relationship between the blood levels of glucose and V.F.A., the relationship is not seen in all cows. Ten cows showed both transient low levels of glucose and transient high levels of V.F.A. soon after calving but the changes were coincident in only five of them. Only 16 normal cows and 5 cows with a history of ketosis were studied, and even within this small group a considerable variation between individual cows was seen. It is therefore probably unwise to make deductions from the results obtained in the present study. The seasonal variation observed in blood glucose, ketone bodies and V.F.A. deserves further study and an attempt made to determine its cause. It is of interest that in three or four cows a tendency was seen to what would usually be regarded as abnormal levels of glucose, ketone bodies and V.F.A., but further study of normal cows in early lactation is needed before it can be decided whether such findings represent the transient beginnings of an abnormal state, or merely the extremes of normal changes. Apart from these few cows, the lesser changes seen in most of the cows studied are worthy of further investigation, to see whether more frequent blood sampling would reveal a sequence of changes. If such studies are to contribute to our understanding of ketosis it is essential to extend Shaw's work by similar observations during the development of ketosis. CONCLUSIONS
Determinations of blood glucose, acetone, acetoacetate and volatile fatty acids were made on 2 I normal cows from 10 weeks before to 10 weeks after calving. In most cows blood glucose increased at parturition and decreased to transient lower levels after parturition. Volatile fatty acid levels were highest in the first few weeks of lactation, and in most cows ketone bodies showed no appreciable change. Seasonal variation in the blood levels of these substances was seen, glucose being significantly lower in winter, volatile fatty acids significantly higher in winter, and ketone bodies lower in winter and early spring. ACKNOWLEDGMENTS
The expenses of this work were defrayed in part by a grant from the Agricultural Research Council. The authors are grateful to Prof. R. A.
D. HORROCKS AND
J.
Y. F. PATERSON
34 1
Morton F.R.S. for his interest in this work; to Prof.]. G. Wright and to Prof. E. G. White for laboratory accommodation at the University of Liverpool Veterinary Field Station; to the clinical staff of the Field Station for their cooperation; and to the managers and dairy staffs of farms associated with the Field Station for their willing and unfailing cooperation. REFERENCES
Allcroft, W. M. (1947). Brit. vet. ].,103,2. Annison, E. F. (1954), Biochem. ]., 58,670. Boddie, G. F. (1935). Vet. Rec., 15, 1939. Conway, E. ]., and Downey, M. (1950). Biochem. ].,47, iv. Eden, A., and Green, H. H. (1940). Vet. Rec., 52, 725. Godden, W., and Allcroft, W. M. (1932). Biochem. ]., 26, 1640. Greenberg, L. A., and Lester, D. (1944). ]. biol. Chem., 154, 177. Knodt, C. B., Shaw,]. C., and White, G. C. (1942).]. dairy Sci., 25,837. Miller, B. F., and van Slyke, D. D. (1936). ]. biol. Chem., 114, 583. Paterson,]. Y. F. (1957). ]. compo Path., 67, 165. Pennington, R.]. (1954), Biochem. ]., 56,410. Sampson, ]., and Boley, L. E. (1945). Cornell Vet., 35, 314. Shaw,]. C. (1943). ]. dairy Sci., 26, 1079. van Soest, P. ]., and Blosser, T. H. (1954), Ibid., 37, 185. [Receivedfor publication, December 22nd, 1956]
COMPo
PATH.
1957. VOL. 67.
33 1
SOME OBSERVATIONS ON GLUCOSE, KETONE BODIES .AND VOLATILE FATTY ACIDS IN THE BLOOD OF DAIRY CATTLE By
D. HORROCKS and ]. Y. F. PATERSON Department qf Biochemistry, University qf Liverpool
INTRODUCTION The determinations of blood glucose, ketone bodies and volatile 'fatty acids described in this paper were carried out simultaneously with the estimations of plasma 17-hydroxycortico-steroids described by Paterson (1957). It was intended to extend observations by ,earlier workers on changes in blood composition at parturition and in early lactation in normal cows.· It has been reported that the blood level of glucose increases just before parturition, decreases immediately after parturition and thereafter is constant for one to four weeks (Godden and Allcroft, 1932; van Soest and Blosser, 1954). Knodt, Shaw and White (1942) and Sampson and Boley (1945) have reported that the blood level of ketone bodies shows no change at parturition, but is higher in early than in late lactation . .No observations on blood levels of volatile fatty acids in pregnancy and lactation appear to have been reported. Shaw (1943) studied the development of ketosis by estimation -of blood glucose and ketone bodies in early lactation in cows from ·a herd with a high incidence of ketosis. In cows developing ketosis the decrease in blood glucose following parturition continued for four to ten days, during which time blood ketone bodies increased markedly, until clinical symptoms of ketosis appeared. It was intended to extend Shaw's work by observations on cows which had a history of ketosis in previous lactations and which might thus be expected to develop ketosis in the lactation under study. None of these animals developed ketosis in the period of study however and so this paper deals only with clinically normal animals. METHODS Blood glucose. Miller and van Slyke (1936). Plasma volatile fatty acids. Conway and Downey (1950). Blood ketone bodies. Blood ketone bodies were estimated by a modification of the method of Greenberg and Lester (1944). The reflux units described by these authors were found inconvenient in routine operation and instead the conversion of ~-hydroxybutyric acid and acetoacetic acid to acetone was carried out in glass-stoppered test tubes heated in a boiling water bath. The stoppers, lubricated by a silicone grease, were retained in the tubes by a rubber-cushioned metal plate. The time of ·extraction of acetone 2:4 dinitro-phenylhydrazone with carbon tetra·chloride was increased to 25 min. since IO min. was found to be in-
332
BIOCHEMICAL EXAMINATION OF THE BLOOD IN CATTLE
sufficient for complete reaction. The 2:4 dinitro-phenylhydrazine· reagent was washed with carbon tetrachloride to give lower blanks as· recommended by Pennington (1954). f3-hydroxybutyric acid was at first estimated by difference from determinations of total ketones and of acetone and acetoacetic acid, but this procedure was eventually found to be unreliable at normal blood levels of these substances due to the inconsistent recovery of acetone and acetoacetic acid in the method for total ketones. The method described by Greenberg and Lester ( 1944) for the direct determination of f3-hydroxybutyric acid was found to be reliable. Unfortunately most of the determinations of f3-hydroxybutyric acid were made by the former method, and therefore no results from estimations of f3-hydroxybutyric acid have been included in this paper. RESULTS
In this and following sections plasma volatile fatty acids will be referred to as plasma V.F.A. The cows in this study were those used by Paterson ( 1957) and since all remained clinically normal throughout the period of study it seemed unlikely that any differences would be found between the normal cows and those with a history of ketosis. in previous lactations. Comparison of Normal Cows and Cows with Previous Ketosis
Since seasonal variations occurred in the blood levels of glucose,. ketone bodies and V.F.A. in normal cows, the results from cows' TABLE
I
COMPARISON OF BLOOD LEVELS
Glucose, ketone bodies and volatile fatty acids in normal cows and cows with previous ketosis Prepartum November-April Cows with previous ketosis Normal cows
Glucose (mg,% blood) Mean ± S,E, (no. of results) Sig. of difference Plasma V.F.A, (mg. acetic acid %) Mean ± S.E. (no. of results) Sig. of difIe rence Acetone and Acetoacetate (mg. acetone % blood) Mean ± S.E. (no. of results) Sig. of difference
Postpartum January-May - - - -Cows with previous ketosis Normal cows
- - - - - - - - - - - - ---- - 43 '5±7'3 (3 7)
44'4±6'I (37)
't' = 0·64, P<0·6
------
7' 2 ±2'4 (38)
7'9±2'5 (40 )
't'= , '28, P <0'3
2'5 ± I'0 (38)
2'O±0'9 (39)
47,6±6'4 4 8 'O±5'4 (45) 1_~9_)_ _ 't' =o '28, P
6'9±3 '0 (44)
6'6±3'7 (59)
' t' = 0 '38, P < o,8
2'4±1'3 (46)
- - - - - - - - - - - - - -- -- 't' = 1'94, P
- - -- -
2'2±"0 (61)
't' = 0'90, P<0'4
D. HORROCKS AND
J.
Y. F. PATERSON
333:
with previous ketosis are compared only with those from normal cows during the same months, November to May. From Table I it can be seen that no differences in the blood levels of glucose,. ketone bodies and V.F.A. were found between normal cows and cows with a history of ketosis in previous lactations. In the following considerations the results from cows with previous ketosis are therefore included with those from normal cows. Changes bifore and after Parturition The mean blood levels of glucose, ketone bodies and V.F.A. for 10 day intervals during the period of study are given in Table 2. The increase in blood glucose between 20 to I I days and 10 to 0 days prepartum is significant ('t'=2·18, P<0.05) as is the decrease between 0 to IO and II to 20 days postpartum ('t'=2·38, P<0.05). No other significant changes in blood glucose level were seen in the period of study. While plasma V.F.A. show a lower level at 0 to 10 days prepartum and a higher level at I I to 20 days postpartum, no significant difference was found between any two successive IO day periods. Postpartum, the mean level of V.F.A. for the period 0 to 30 days (7· I ±3· 3 mg. per cent) is significantly higher than the mean level for the period 31 to 70 days (5·9 ±3·0 mg. per cent), 't' =2·63, P< 0·01. No significant changes occurred at any time in the blood level of ketone bodies. Thus the overall picture is that the blood level of glucose increases just before parturition, and decreases thereafter; plasma V.F.A. levels are slightly higher in the first few weeks of lactation than subsequently, and ketone body levels show no change in the first 10 weeks of lactation. However, while most animals showed such a picture, there was some variation among individuals~ particularly in the postpartum period. The results for four individual animals (Fig. I) show the extent of this variability. Of the 2 I cows used in this study 14 showed an increase in blood glucose before calving followed by a decrease soon after calving (examples A, B and D, Fig. I). Four cows showed no apparent change in blood glucose over the period of parturition (example C). There was some variation in the extent of the decrease in blood glucose after calving and in the constancy of the postpartum glucose level. Of the 14 cows showing a decrease in blood glucose soon after calving, 8 showed a decrease to levels below 45 mg. per cent glucose, in some cases the decrease being very striking as in example A. Following these lower levels there was an increase to a relatively constant level. In others, after the decrease at parturition, the blood level of glucose was constant (example B) or increased slightly. Of the 7 cows which showed no appreciable change in blood glucose around parturition 6 showed a transient decrease to rather low levels 3 to 5weeks after calving (example C). Thus two-thirds of the individual
Mean ± S.E, (no, resu lts)
.
( 12)
2'O ±
I 'I
6'8 ± I'7 ( 12)
Plasma VFA (mg, acetate %) Mean ± S,E, (n o, results)
Acetone and acetoac etate (mg. acetone %)
46 '6±6 '3 ( II )
Glucose (mg, % blood) Mean ± S,E, (no, results)
70.6,
2' 1 ±o'7 ( 12)
6'6 ± I'5 ( 12)
45 '4 ± 4'5 (12)
60-51
TABLE 2
o' l± o'g ( 15)
7'O±2'3 ( 15)
44'2±4'7 (13)
2'g ± I ' 2 (2 1)
7'3 ± 3'2 (21)
42'8 ± 4 '8 (21)
----
.
2'7 ± I ' 1 ( Ig)
(20)
-
6'B±2'8
44'5±7' 7 ( 18)
2 '4 ± I 'o (21)
6'5±3'4 ( Ig)
46 'I±6'I (18)
20-II
2'7 ± 1'0 (27)
5'6±3'O (25)
52'4 ±8 '6 (27)
10-0
2 '7± "1 (3 2)
6'7 ± 2'6 (3 1)
5 I'7± 9 '8 (30)
0-10
3'2±2'6 (2g)
8'2±3'9 (27)
46 '1 ±5'g (2g)
11-20
2,6±I '3 (33)
6'6 ± 2 'g (32)
45'8±7'8 (32)
21-30
2'6± I 'O (23)
5'4±2'6 (24)
4 8 'I ± 5" (24)
4I -50
5,-60
2'5 ±I' 7 (23)
5 'g ±2'6 (23)
2'3± I 'O (2 1)
6'7±3'7 ( 17)
50'3 ± 16,8 48'1 ±4'2 (22) (2 1)
3 '-4 0
30 - 21
4 0-3'
50-4 l
D ays after parturition
D ays hefore parluriliQn
Clucose, ketone bodies and volatile fatty acids during pregnancy and lactation
CH ANGES I N THE BLOOD LEVELS
2'8± I' 7 (21)
5'7 ± 3'3 ( Ig)
48'4 ± 7'I (20)
6'-7°
~
C'l
Z
oI::)
o
t>:I t:D t'"
:J:
..,
o z o..,
~
~
t'"
C'l
I
t:D
o
~
t.>:>
(..>0
D. HORROCKS AND
J.
335
Y. F. PATERSON
cows showed a transient decrease in blood glucose to a moderately low or low level in the first ten weeks of lactation. Fig.
I.
FIGURE I EXAMPLES OF RESULTS FROM INDIVIDUAL COws 00
GLUCOSE
::E~
20
20V I
12
12
40
... 0-%
-
.V FA
KETONES
VFA
~VFA MG.%
"......... - ~ --~ . -------!-.. 50
GLUCOSE
MG%
[----+____ "__...
0 _____ 0 _0_ _ 0
30t:
I
,--./
70
so
C
D 12
KETONES &VJA.
_. . . . .1-.....___
... 0%
~
~
0
~
w
ro
~ m ~ ro ~ ~ 0 ~ w DAYS BEFORE OR AFTER PARTURITION
0 ' · - - -. •
ro
~
~
~
About half of the cows showed a decrease in the plasma level of V.F.A. before parturition. After calving, half of the cows showed transient high levels ofV.F.A. (examples Band C, Fig. r). Five of these showed a fairly well defined peak in V.F.A. (example B) but in six there was much fluctuation in the V.F.A. level (example C). Ten of these eleven cows showing high levels of V.F.A. postpartum showed also transient decreases in blood glucose; in six cases the lowest level of glucose was approximately coincident with the highest level of V.F.A., as in example C. Four cows showed a general increase in plasma V.F.A. throughout the postpartum period studied (example A) and four showed a general decrease (example D). Only six cows showed any appreciable change in the blood level of ketone bodies, and these showed transient high levels approximately coincident with a high level of plasma V.F.A. postpartum (example C). No pattern of agreement between changes in blood levels of these substances and changes in milk yield or the level of milk yield could be seen. Seasonal Variation The results showing seasonal VarIatIOn in the blood levels of glucose, ketone bodies and V.F.A. are given in Table 3. Both prepartum and postpartum the blood level of glucose was significantly higher in the period June to September than in other periods.
c
~
336
BIOCHEMICAL EXAMINATION OF THE BLOOD IN CATTLE
When considered by calendar months (Fig. 2), blood glucose is seen to be highest in July, to decrease progressively until December and then to increase slightly between January and March. Fig.
q
2.
-FA
---J(B
----
-
I--
r-
zc( ~~50 ~
III
840 3
"
30
1--1--
r-
- -
-
Jy Au sP· Oc No DE JA Fe MR AP My MONTH
FIG. 2 : SEASONAL VARIATION I N BLOOD COMPOSITION
The plasma level ofV.F.A. prepartum was significantly lower in the period June to September than in the period October to December, and was lower in this period than in January to April. From Table 2 it would appear that the plasma level ofV.F.A. postpartum was no different for the period July to September than for the period October to December. However among the cows studied in the period July to September was one which, although remaining clinically normal, showed very high levels of ketone bodies (14 mg. per cent) and V.F.A. (17 mg. per cent) for a short time. If the results from this animal are omitted from the results for the period July to September, the mean 'plasma level of V.F.A. is decreased to 4'9 ±2·3 mg. per cent and while this is not significantly different from the mean for the period October to December, the overall postpartum picture now resembles more closely the prepartum picture. From Fig. 2 it can be seen that plasma V.F.A. levels are highest in the period,December to March, i.e. approximately when blood glucose levels are lowest.
't'=O'2 7,
Sig, of difference
2'9±I'5 (4:»
I
6'4±2'7 (49)
[
2 'O±O '9 (53)
't'=2'52, P
7'6±2'S (53)
't'=o'59, P < o,6
46 'o±8,o (50)
't'=4'73, P
45'o± 10 '2 (48 )
P<~
't'=2,64, P < O'02
Sig, of difference
2'g±I'2 (20)
4'5±2'4 (23)
Plasma VFA (mg, acetic acid %) M ean ± S,E, (no, of results)
Acetone and Acetoacetate (mg, acetone % blood) Mean ± S,E, (no, of results)
't' = 2,6" P < O'02
5 "4± 8'o (23)
't'=2'27, P
3'g±2'5 (32)
't'=O'20, P
6'1±3'8 (3 0 )
I
I
3'O±O'7 (45)
I
5'9±2'4 (49)
I
4 6 '8±8'4 (51)
't'=3,6g, P
54'2±g'4 (30)
47'1±6'1 (92)
January-May
7'3±3 '2 (92)
I
't'=3 '40, P
2'2 ± I" (g6)
't'=2'79, P
I
't'=O'22, P
October-December
July-September
October-December
January-April
Postpartum
Prepartum
Glucose, ketone bodies and volatile fatty acids,
Sig, of difference
Glucose (mg, % blood) Mean ± S,E, (no, of results)
June-September
TABLE 3 SEASONAL VARIATION IN THE BLOOD LEVELS
-
-
!::l
-..J
c..J c..J
~
~ ~
!'l
:<
':-'
t1
Z
>
g;
oC'l
~
~
o
:I:
338
BIOCHEMICAL EXAMINATION OF THE BLOOD IN CATTLE
The prepartum blood level of ketone bodies was higher in the period June to December than in the period January to April (Table 3). The postpartum level was higher in July to September than in other periods. If the results from the cow showing rather higher than usual levels of ketone bodies and V.F.A., are excluded from the total results for this period, the mean for this period becomes 3.2 ±1·4 mg. per cent which is little different from the mean for the period October to December. From Fig. 2 it can be seen that blood ketone body levels are lowest in the period January to April. DISCUSSION
The determinations of plasma volatile fatty acids described in this paper represent an extension of observations by earlier workers on the composition of bovine blood in pregnancy and lactation. The method used gives good recoveries of formic, acetic and propionic acids which, according to Annison (1954), are the volatile fatty acids present in bovine plasma. It was not possible in the present work to confirm the identity of the acids estimated, but it is hoped to do so in subsequent work. Annison has found that of the total V.F.A. in bovine plasma 90 per cent is acetic acid and the values found by him (0·8 mM V.F.A. per litre=4·8 mg. per cent acetic acid) are not greatly different from the mean value in the present work (6.6 mg. per cent acetic acid) although in the present work the normal range was found to be quite large (I to 17 mg. per cent). The seasonal variation in the blood levels of glucose, ketone bodies and V.F.A. in normal cows is of interest. A seasonal variation in the blood level of ketone bodies has been reported by Sampson and Boley (1945) who found higher levels in June and July than in November to January. These workers observed their animals only during these two periods whereas in the present work the period of observation was continuous from June to May. Our finding that blood ketone body levels are lowest in the months January to April is therefore not in conflict with the finding of Sampson and Boley. In contrast Knodt et al. (1942) have reported that blood ketone body levels are highest in winter, but they considered that the increase was due primarily to winter feeding of silage to the animals studied. Eden and Green (1940) reported that blood ketone body levels in a herd of 60 cows were higher in March than in July, a finding contrary to that in the present work and in the work of Sampson and Boley (1945). It is unfortunate that the ketone body levels reported in the present paper comprise only acetone and acetoacetic acid, but it is considered unlikely that the omission of ,8-hydroxybutyric acid would account for the discrepancy with the findings of Eden and Green. No explanation can be offered for this variation in ketone body levels which is contrary to what might be expected from the seasonal
D. HORROCKS AND
J.
Y. F. PATERSON
339
variation in blood glucose levels. That the blood level of glucose is lowest in winter (December and January) is in accord with the seasonal incidence of ketosis in this country, most cases occurring in the winter months (Allcroft, 1947). While the aetiology of ketosis is in some respects still obscure it is clear that the development of a hypoglycaemic condition is one stage in the development of ketosis, and it is possible that the low winter levels of glucose observed in this work indicate a tendency to the development of hypoglycaemia. It is unlikely that the seasonal change in blood glucose is related to the seasonal change in plasma 17-hydroxycorticosteroids reported previously (Paterson, 1957), for while the maximum blood level of glucose coincides with the maximum level of steroid in June and July, there the apparent correlation ends. There may be a relationship between the lower blood glucose in winter and the higher level ofV.F.A. at that time, although there is nothing in the present work to show that one is the consequence of the other. An increase in blood glucose before calving, and a decrease after calving were first reported by Godden and AIlcroft (1932), and have subsequently been reported by Shaw (1943) and by van Soest and Blosser (1945). From the results given by Shaw the postpartum decrease in blood glucose can lead to transient lower levels of about 40 mg. per cent glucose, and from the results of van Soest and Blosser transient lower levels of 36 to 44 mg. per cent may be found soon after parturition. In the present work an increase in blood glucose level at parturition was seen in two thirds of the cows studied, and in the same proportion of animals there were seen also decreases postpartum to transient low levels of glucose, these lower levels ranging from 23 to 45 mg. per cent glucose. The high levels of blood glucose at parturition may be related to the increase in plasma I7-hydroxycorticosteroids at that time (Paterson, 1957) for in most cows changes in blood glucose were parallel to changes in corticosteroids. Boddie (1935) however, detected lactose in the urine of parturient cows and suggested that the increased level of blood sugar at calving might be due to resorption of lactose from the udder. No explanation can be offered for the transient low levels of glucose postpartum observed in the present work and by earlier workers. The rate and extent of the postpartum decrease in blood glucose in normal cows are, however, considerably less than in those cows developing ketosis observed by Shaw (1943). He found that the decrease in blood glucose just after parturition continued for 4 to IO days, the final levels being 15 to 20 mg. per cent glucose, but in the present work a similar decrease was seen in only one cow (example A, Fig. I) whose blood glucose level decreased from 57 mg. per cent to 29 mg. per cent in one week. In the present work only three cows showed transient higher ketone body levels (5 to 6 mg. per cent acetone) associated with lower blood glucose levels. Shaw found that during the development
340
BIOCHEMICAL EXAMINATION OF THE BLOOD IN CATTLE
of a marked hypoglycaemia blood ketone body levels increased rapidly until clinical symptoms ofketosis appeared. It would appear that in most normal cows ketone body levels undergo no appreciable change. The results presented in Table 2 and in Table 3 suggest that changes in blood glucose level might be reflected to some extent by changes in plasma V.F.A. level. In a few cases of ketosis where there was an opportunity for blood examination it was found that very high plasma levels of V.F.A. were associated with hypoglycaemia and ketonaemia (unpublished results). However while there might be some general relationship between the blood levels of glucose and V.F.A., the relationship is not seen in all cows. Ten cows showed both transient low levels of glucose and transient high levels of V.F.A. soon after calving but the changes were coincident in only five of them. Only 16 normal cows and 5 cows with a history of ketosis were studied, and even within this small group a considerable variation between individual cows was seen. It is therefore probably unwise to make deductions from the results obtained in the present study. The seasonal variation observed in blood glucose, ketone bodies and V.F.A. deserves further study and an attempt made to determine its cause. It is of interest that in three or four cows a tendency was seen to what would usually be regarded as abnormal levels of glucose, ketone bodies and V.F.A., but further study of normal cows in early lactation is needed before it can be decided whether such findings represent the transient beginnings of an abnormal state, or merely the extremes of normal changes. Apart from these few cows, the lesser changes seen in most of the cows studied are worthy of further investigation, to see whether more frequent blood sampling would reveal a sequence of changes. If such studies are to contribute to our understanding of ketosis it is essential to extend Shaw's work by similar observations during the development of ketosis. CONCLUSIONS
Determinations of blood glucose, acetone, acetoacetate and volatile fatty acids were made on 2 I normal cows from 10 weeks before to 10 weeks after calving. In most cows blood glucose increased at parturition and decreased to transient lower levels after parturition. Volatile fatty acid levels were highest in the first few weeks of lactation, and in most cows ketone bodies showed no appreciable change. Seasonal variation in the blood levels of these substances was seen, glucose being significantly lower in winter, volatile fatty acids significantly higher in winter, and ketone bodies lower in winter and early spring. ACKNOWLEDGMENTS
The expenses of this work were defrayed in part by a grant from the Agricultural Research Council. The authors are grateful to Prof. R. A.
D. HORROCKS AND
J.
Y. F. PATERSON
34 1
Morton F.R.S. for his interest in this work; to Prof.]. G. Wright and to Prof. E. G. White for laboratory accommodation at the University of Liverpool Veterinary Field Station; to the clinical staff of the Field Station for their cooperation; and to the managers and dairy staffs of farms associated with the Field Station for their willing and unfailing cooperation. REFERENCES
Allcroft, W. M. (1947). Brit. vet. ].,103,2. Annison, E. F. (1954), Biochem. ]., 58,670. Boddie, G. F. (1935). Vet. Rec., 15, 1939. Conway, E. ]., and Downey, M. (1950). Biochem. ].,47, iv. Eden, A., and Green, H. H. (1940). Vet. Rec., 52, 725. Godden, W., and Allcroft, W. M. (1932). Biochem. ]., 26, 1640. Greenberg, L. A., and Lester, D. (1944). ]. biol. Chem., 154, 177. Knodt, C. B., Shaw,]. C., and White, G. C. (1942).]. dairy Sci., 25,837. Miller, B. F., and van Slyke, D. D. (1936). ]. biol. Chem., 114, 583. Paterson,]. Y. F. (1957). ]. compo Path., 67, 165. Pennington, R.]. (1954), Biochem. ]., 56,410. Sampson, ]., and Boley, L. E. (1945). Cornell Vet., 35, 314. Shaw,]. C. (1943). ]. dairy Sci., 26, 1079. van Soest, P. ]., and Blosser, T. H. (1954), Ibid., 37, 185. [Receivedfor publication, December 22nd, 1956]