Origin of Plasma Fatty Acids in Lactating Cows Fed High Grain or High Fat Diets

Origin of Plasma Fatty Acids in Lactating Cows Fed High Grain or High Fat Diets D. L. PALMQUIST and H. R. CONRAD

Department of Dairy Science Ohio Agricultural Research and Development Center, Wooster 44691

the mammary gland from the plasma, due to reduced mobilization of fatty acids from adipose tissue. Glaseock et a]. (10) fed 9,10 tritiated stearie acid to goats and a cow and followed the time course of secretion of radioactivity in. milk fat. F r o m empirical ealeulations, they estimated that 25% of the milk fat was derived directly from dietary fat. Sinee the long-chain fatty acids provide about 50% of the milk fat mass (8), the question of origin of plasma f a t t y acids seemed worthy of investigation.

Abstract

Palmitate-l-14C was given to lactating cows in 20 trials involving four dietary treatments and two different routes of tracer administration. The specific activitytime curve of 14-C' activity in milk fat was resolved by curve analysis into two components, a rapid-turnover component attributed to exogenous (dietary) fatty acid and a slower turnover component attributed to adipose tissue. Administration of palmitate-lJ4C abomasally or orally gave greater (P < 0.01) estimates of exogenous fatty acid than did intravenous dosing. A high grain-restricted roughage diet reduced (P < 0.01) the exogenous estimate, presunmbly due to increased uptake of dietary fatty acid by adipose tissue. A low-fat diet also lowered the estimate of exogenous fatty acid. A high-fat diet reduced the turnover time of the adipose tissue pool by 30% whereas the high grain restricted roughage diet increased the turnover time by 26%. Estimates of the effects of dietary treatments on rate constants of fatty acid transfer in a 2-pool model are presented. Low fat and high grain restricted roughage diets reduced plasma free fatty acids (P < 0.01 and 0201). The high grain restricted roug]hage diet increased plasma glucose and serum heparin-preeipitable lipoprotein esters (P < 0.01 and 0.001).

Materials and Methods

Introduction

Diets which induce low milk fat content are precisely those which cause greatest adipose tissue deposition (26). This observation was complemented by Opstvedt et al. (20), who showed greater enzymatic adaptation to milk fat-depressing diets by adipose tissue than by mammary tissue. Yah Soest (26) proposed that the cause of low milk fat may be a deficiency of long-chain fatty acids provided to Received for publication January 6, 1971. Journal Article 113-70, Ohio Agricultural Research and Development Center, Wooster 44691. 1025

The approach to the problem was to feed or inject directly into the jugular vein a pulse dose of palmitate-l-14C, follow the secretion of labeled palmitate into milk fat, and partition the specific actlvity-time curve into dietary and adipose component pools by curve analysis. Dietary fat and h a y : g r a i n ratios were the variables. Some characteristics of cows, diets, and production are in Table :1. Cows in Trials 1 to 20 were Jerseys and Guernseys. Those in Trials 22 to 27 were Holsteins. Wide ranges of stages of lactation, hay-concentrate ratio, dietary fat, and production were represented. Milk fat production ranged from 100 to 1,500 g per day. Cows were housed in metabolism stalls where radioactive excreta could be collected for safe disposal. They were fed twice daily, and orts were removed once daily. Cows were conditioned to the diets prior to initiation of trials, and polyethylene catheters (Clay-Adams, P E 200) were placed bilaterally in the external .jugular veins the day preceding each trial. The 1-14C pahnitate dose was given per abomasum in Trials 1 and 2, per os in Trials 3 and 4, and via the jugular catheter in all other trials. Oxytocin (10 units) was injected intravenously to assure complete removal of milk before dosing and at 3, 6, 9, 12, 18, 24, 32, 40 and 48 hr after dosing. The cows were then milked without oxytoein at approximately 10- and 14-hr intervals for the duration of the ]0-day trials. Milk samples at each milking were tested for

1026

P A L M Q U I S T AND CONRAD

TABLg 1. Stage of lactation, diet, and production of cows used to study plasma fatty acid pools.

Trial

Stage of lactation

Dietary treatment a

(days) 1 2 3 4 5 6 7 8 15 16 17 18 19 20 22 23 24 25 26 27

300 15 240 54 18 27 40 48 40 170 190 135 144 154 220 200 75 270 35 140

CG CG CG CG C C C C LF LF LF LF HF HF HGRR b ttGRR HGRR ttGRR C C

Dietary ether extract

Milk production

Milk fat

Milk fat

(g/day)

(kg/day)

(%)

(g/day)

464 236 490 637 662 547 532 411 215 270 163 170 1,530 1,289 320 512 560 424 429 416

6.13 16.34 10.44 22.25 29.28 17.80 30.32 15.26 17.30 9.39 6.32 3.54 14.17 22.33 17.01 16.53 22.50 13.39 29.07 15.15

7.2 9.0 5.6 5.3 5.2 3.8 3.7 3.9 6.0 4.8 6.1 9.8 4.5 3.9 3.7 0.9 0.7 0.8 3.8 2.6

441 1,470 585 1,178 1,525 677 910 595 1,042 451 386 348 638 878 625 150 155 104 1,120 391

a Dietary treatments : CG : Control diet, palmitate-l-14C dose given orally or abomasally; C : Control diet, palmitate-l-14C dose given intravenously; L F : Low fat diet; t t F : High fat diet; H G R R : High grain-restricted roughage diet. b Data were included with the control group, as the cow did not respond typically to the HGRR diet. f a t - p e r c e n t a g e by the Babcock procedure. F i f t y to 100 mg of the fat were transferred from the Babcock bottles to liquid scintillation vials, dissolved in 10 ml scintillation fluid (5.0 g PPO and 300 mg P O P O P / l i t e r of toluene) and counted in a Packard Model 314 E X liquid scintillation spectrometer. Counts were corrected to distintegrations by adding 14C-toluene as an internal standard. Specific activity of the milk fat is expressed as disintegrations per minute per gram. Daily dry matter intake was recorded. Samples of feed and refusal were taken for analysis of total ether extract by Soxhlet extraction and total nitrogen by the Kjeldahl method. Plasma free fatty acids were determined titrimetrically after extraction as described by Annison (1). tteparin-precipitable lipoproteins were prepared by scaling up fivefold the procedure o£ Huber et al. (17). The pellet was isolated by centrifngation at 1000 X g and total esters were extracted from the lipoJOURI~AL OF DAIRY SCIENCE ~OL. 54, NO. 7

proteins according to Folch et al. (7) and assayed as their hydroxamates according to Palmquist et al. (22). Plasma glucose was assayed by a commercial glucose oxidase preparation (Worthington Biochemical Corp., Freehold, N. J.) on Somogyi filtrates. Samples of tureen content were taken through rumen fistula or by stomach tube. Approximately 50 g of rumen contents were added to a tared bottle containing 10 ml of 10 ~ tI4PO 4. The exact weight of the rumen contents was determined, the sample was diluted to two times the original volume with distilled water, and the samples were stored at 4 C. This approach is based on the assumption that the density of rumen contents is not significantly different from 1.0. After equilibration of the aqueous phase with the rumen content, samples were centrifuged at 35,000 X g. Rumen volatile fatty acids in the supernatant were quantitated by comparison with authentic standards in an F and M Model 402 gas-liquid chromatograph with a 4 mm id X 120 cm glass column packed

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O R I G I N OF P L A S M A F A T T Y A C I D S

with 10% Carbowax-20 M-terephthalic acid on 60/80 mesh acid-washed Chromosorb W. Means and standard error were by least squares analysis of variance described by Harvey (16). Treatment mean differences were tested by t. The pahuitic acid-l-14C dose (International Chemical and Nuclear Corp., Irvine, California) was absorbed on corn meal and placed in a gelatin capsule for oral or abomasal doses. For intravenous injection, it was adsorbed on bovine serum albumin, dissolved in 0.10 K H P O 4 buffer, p H 7.0, and sonicated to assure complete solution. Ten to 15 ml of solution, 10 to 20 ~Ci/ml, were injected in each trial. I n Trials 1 and 2, two rumen-fistulated Jerseys, fed concentrates and corn silage, were given 400 tLCi of palmitate-l-14C into the abomasum via the tureen fistula. Conditions were changed for Trials 3 and 4 in which two Jerseys fed hay, concentrates and corn silage were given 400 t~Ci of palmitate-l-14C absorbed on ground corn in weighted gelatin capsules per os. The cows were fed alfalfa hay and concentrates in Trials 5 and 6. Two-hundred-fifty t~Ci of palmitate-l-14C were injected via the jugular vein. The same cows and diets were used for Trials 7 and 8 as for Trials 5 and 6 after 30 days were allowed for elimination of most of the radioactive carbon from the fat depots. I n Trials 7 and 8 glucose (Cerelose, Nutritional Biochemicals Corporation) was infused continuously into the jugular vein o£ each cow with a Sigmamotor peristaltic pump at 2.4 megaeal per day during the 10 days. A semi-synthetic low-fat diet was fed in Trials 15 to 18 and compared with a semisynthetic high-fat diet fed in Trials :19 and 20. Ingredients in the low- and high-fat diets are in Table 2. I n Trials 22 to 25, a pelleted grain concentrate was fed ad libitum with restricted roughage and compared with a normal diet containing alfalfa hay, corn silage, and grain concentrate in Trials 26 and 27.

TABLE 2. Composition of semi-synthetic diets. Low fat

Component

(%) Cane molasses Dried beet pulp Ground barley Solvent extracted soybean oilmeal Hydrogenated edible fat Diealcium phosphate Salt Vitamin A, I U / k g Vitamin D, I U / k g Ether Dry Crude Dry

High fat

(%)

5.0 60.0 20.0

-57.0 20.0

13.0 -1.0 1.0 2,200 1,000

13.0 8.0 1.0 1.0 2,200 1,000

extract, % of matter protein, % of matter

1.76 15.3

11.2 14.0

Results and Discussion

For statistical treatment, data were arranged and analyzed according to the responses of the cows. As the major characteristics were not significantly influenced by glucose infusion in Trials 7 and 8 (P > 0.05), these data were included with control diet data. I n Trial 22, milk fat depression was not accomplished, and the rumen volatile fatty acids were not changed from control values so that the data were included with those from the nondepressed group. Rumen fermentation patterns, as reflected by volatile fatty acid proportions, are in Table 3. Although measurements were not made in all experiments, all experimental diets are represented. Both control and high-grain, restricted roughage diets were fermented in typical fashion as shown by the acetate-propionate ratios. The rumen fluids from semi-synthetic low- and high-fat diets, although within the range of normal volatile fatty acid patterns, were relatively higher in butyrate, possibly

TABLE 3. Mean molar percentages of the rumen volatile fatty acids. Dietary treatment a

Acetic

Propionic

C HGRR LF HF

66.7 49.4 61.3 65.4

17.0 39.0 18.6 18.3

n-Butyric (molar %) 9.9 5.4 16.5 12.8

iso-Valeric

n-Valeric

4.4 0.7 1.1 1.1

1.8 4.9 1.9 2.0

Ac/Pr 3.92 1.26 3.30 3.57

a See Table 1. JOURNAL

0~ D A I R Y

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XTOL. 54, NO, 7

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PALMQUIST

reflecting the large (60%) proportion of beet pulp in the diet (27) which is mostly highly digestible cellulose and po]yuronides. A typical semi-Iog plot of the time-excretion curve of 14C activity into milk fat is in Figure 1. A logarithmic plot (base ]0) of the specific activity-time curve of milk fat resulted in a non-linear decay curve indicating that palmitate was passing through more than one pool, each with a different turnover rate, before being transferred to milk fat. Visual inspection of the curves showed no differences in shape due to route of palmitate-l-14C administration or type of diet. Route of administration did affect time of maximal specific activity in milk fat (gut, ]8 to 24 hr; intravenously 9 to 12 hr). The time of maximum specific activity is in agreement with the earlier data of Glascock et al. (10). Analysis by curve peeling (6, 23) showed that the milk fat specific activity curve could be separated into two straight line decay curves, one with a steep slope (rapid turnover rate), and one with a relatively flat slope and long

AND CONRAD

half-life. The contribution of each pool to the total plasma fatty acid was assessed as follows : 0.63212 Y% "gp = Total DPM excreted from pool p during one turnover time of the pool. Turnover time = 0.693/2.3 × slope p Y% : extrapolated intercept of the Y axis of slope p ( D P ~ / g fat). 0.63212 = 1 -- e-1 : integral of a negative slope to obtain the area under the curve for one turnover time. g, : grams of milk fat secreted during one turnover time of pool p. The contribution of each pool is expressed as a percentage of the sum of the two pools. The rapid turnover pool was arbitrarily designated as exogenous (dietary) fatty acid; the pool with slower turnover rate was designated as adipose tissue. A summary of some of the kinetic measures is in Table 4. The most striking difference between estimates of dietary contribution to

.........

~ 10~

\

HOURS Fro. 1. Typical pattern of radioactivity in milk fat after giving palmitate-l-14C intravenously. Closed circles: observed data points; Crosses: data calculated by curve peeling (6, 23); - 14C excretion curve; . . . . . . . . : extrapolation of slower turnover component to zero time; . . . . . . : rapid turnover component. JOURNAL OF DAIRY SCIENCE VOL. 54, NO. 7

TABLE 4. Summary of least squares analysis of kinetic characteristics.

Dietary treatment a

No. of trials

Exogenous estimate ( % 0 2 plasma fatty acid) 92.6 b ±2.86

Fractional excretion

Turnover time, exogenous pool

Turnover time, endogenous pool

(%) 48.2 ±3.54

(hr) 12.8 ±2.62

(hr) 80.6 ±1.90

.0756 ±.00956

KA

K BA

KAA

.00329 ±.00263

.0135 ±.00114

.0788 --+---.0112

KAB

o

CG

4

C

7

68.8 --+1.74

60.7 ±2,15

12.6 -+-1.59

86.6 ±1.15

.0540 ±.00580

.0243 ±.00160

.0188 ±.000692

.9783 ±.00677

ItGRR

3

51.6 ±3.33

39.3 ±4.06

16.9 ±3.00

109 ±2.18

.0291 ±.0110

.0213 ±.00302

.0171 ±.00131

.0505 ±.0128

LF

4

54.6 ±2.86

43.5 ±3.54

12.9 ±2.62

91.2 ±1.90

.0572 ±.00956

.0185 ±.00263

.0164 ±.00114

.0755 ±.0112

HF

2

69.3 _+3.66

69.8 ±4.53

7.81 ±3.34

60.4 ±2.43

.0773 ±.0122

.0280 ±.00336

.0234 ±.00146

.105 ±.0142

P < .01 NS P < .09

NS NS P < .02

NS P < .06 NS

Summary of test of significance o

CG vs. C C vs. H G R R o~ L F vs. I-IF

-4

df 9 8 4

P < .01 P < .01 P < .05

P < .02 P < .01 P < .02

NS NS NS

P < .02 P < .01 P < .01

P < .08 P < .07 NS

a See Table 1. b Least squares mean ± standard error of the mean.

~a 9 .-.1

tO

1030

PALMQUIST AND

milk fatty acids was due to route of administration of the tracer dose (orally or abomasally versus intravenously, P < 0.01). This may reflect a difference in metabolism between chylomicrons and lipoproteins. Chylomicra, synthesized by the gut wall, are cleared very rapidly by mammary tissue (15, 18). It is possible that significant amounts of the palmitate-l-14C given via the gut were cleared by mammary tisue before they reached equilibrium in the plasma. Very low density lipoproteins and low density lipoproteins are metabolically the most active lipoproteins synthesized by the liver (3, 11, 13). Quantitatively, the greatest portion of the plasma lipoprotein triglyeerides are in the very low density lipoproteins (Palmquist, 1971, unpublished observations). Thus, the difference between the two routes of administration in estimates of exogenous fatty acids could be explained by distribution of the labeled palmitate in pools differing greatly in size, or, alternatively if mammary tissue had a relatively lower affinity for the very low and low density lipoproteins than ehylomicra. When a high grain-restricted roughage diet was fed, the estimate of dietary contribution to milk fatty acids was lower than controls (P < 0.01). Since the quantities of fatty acid in the diets were not different, these data suggest that transport of dietary fatty acid directly to milk fat was reduced by the high grain-restricted roughage diet, probably due to increased uptake of the dietary fatty acids by adipose tissue. This suggestion is supported by the report of Benson et al. (4) that adipose tissue lipoprotein lipase and glyceride synthetase activities were markedly increased when a high grain-restricted roughage diet was fed whereas these enzymes in mammary gland were unaffected by diet. Thus, the conclusion that high grain-restricted roughage diets increase adipose tissue uptake of dietary fatty acids broadens the glucogenic postulate of Van Soest that depressed milk fat may be due to reduced release of fatty acids from adipose tissue (26). The estimate of exogenous contribution of fatty acids to milk fat was reduced (P < 0.05) when a low-fat diet was compared to a highfat diet. An additional purpose of feeding the low-fat diet was to determine whether there was sufficient fatty acid in the diet to satisfy the dietary estimate from integrating the specific activity-time curve. This requirement was met in every case. Although there was a tendency toward reduced milk fat production when the low-fat diet was fed, it did not approach significance. Storry et al. also observed JOURI~AL OF DAIRY SCI~lqCE ~Y~0L. 54, NO. 7

CONRAD

that feeding a low-fat diet (170 g/day) did not reduce milk fat output, but that increased dietary fat did increase milk fat production (24, 25). Diet affected significantly the fractional rate of 14C excretion. When the control diets were fed, 61% of the total ~4C excreted in milk fat during the 10-day trials appeared in milk fat during the first 24 hr. This was reduced by one-third, to 39% (P < 0.01) when the milk fat depressing diet was fed. Varying dietary fat also influenced the rate of 1~C excretion into milk fat (P < 0.02). The turnover times of exogenous pools, although ranging by twofold among diets, were too variable to be affected significantly by diet. However, turnover times of the endogenous pool (adipose tissue) were influenced significantly by diet. Turnover time of the endogenous pool when a control diet was fed was 87 hr; this was increased to 109 hr (P < 0.01) in cows fed the milk fat depressing diet. The high-fat diet reduced the turnover time of the endogenous pool (P < 0.01). The zero time intercepts and slopes of the 14C excretion curves were used to calculate specific kinetic data according to case l a described by Gurpide et al. (14). This model, shown in Figure 2, assumes that K B ---- 0; that is, there is no terminal utilization of fatty acid within pool B, and, thus, all loss of fatty acid from pool B is through pool A, described by ]~BA. The model has been successfully applied in studies of cholesterol turnover (12, 19). Pool A is blood plasma; the dietary fatty acid absorbed into plasma is represented by SA; and transfer of fat from plasma to the mammary gland is represented by KA. Pool B is adipose tissue; SB represents synthesis of fatty acid within the adipose tissue; KAB and KBA represent transfer of fatty acid from plasma to adipose tissue and mobilization of fatty acid, respectively. Few of the rate constants generated were influenced significantly by composition of the diet, a result associated with small differences between means and a large degree of variation. I t may be that the highly dynamic metabolism of free fatty acids and low density lipoproteins in the cows does not permit valid estimates of these kinetic measures. The aforementioned evidence for increased deposition of dietary fatty acid into adipose tissue is not supported by an increase in the magnitude of KAB. However, a comparison of control and milk fat depressing diets, where differences between other measures are most readily explained, shows a 50% reduction in KA, the rate constant of transfer

ORIGIN

A

OF PLASMA

KAB .J

KBA

B

SKA"

FIG. 2. General two-pool (A and B) model. Rate constants are denoted by the K values; SA and SB are rates of entry of material into the pools (12, 14). of fatty acid from plasma to the mammary gland. An increase (P < 0.02) in K~A (high fat versus low f a t diets) supports the observation of increased turnover rate of adipose tissue when the high fat diet was fed. Application of the same data (intercepts and slopes) to formulas developed by Gel]horn et al. (9) for estimation of relative sizes of sodium pools, did not yield data which were physiologically applicable. Multiple regression analysis showed that, in contrast to reports of Storry et al. (24, 25), neither the quantity of dietary ether extract nor the percent of milk fat influenced significantly the estimate of the contribution of exogenous f a t to milk fat. The most valid prediction of the exogenous estimate was from the quantity of milk fat produced; thus, Y = 46.6 + 0.0229X; S~ • x = 2.35; r =

1031

FATTY ACIDS

0.725 (P = < 0.01), where Y = per cent exogenous fatty acid, and X ---- grams milk fat produced per day. The data did not include those from gut infusion trials. I n Table 5 are data on plasma glucose, free fatty acids, and heparin-preeipitable lipoprotein total esters, from experiments with Holsteins (Trials 22 to 27) involving 3 experiments with a control diet and 3 with a milk fat depressing diet. Plasma free fatty acids were decreased 50% by the high grain-restricted roughage diet (P < 0.001) whereas heparinprecipitable lipoprotein esters were increased 80% (P < 0.001). Mean plasma glucose concentration was increased 10% by the high grainrestricted roughage diet (P < 0.01). The low fat diet reduced plasma free fatty acids by 36% as compared to the high fat diet (P < 0.01) in contrast to the data of Storry et al. (24), who showed a reduction in all plasma lipid classes except free f a t t y acids when a low fat diet was fed. The results of this research establish a minimum of two pools of milk fatty acids derived from blood plasma. However, the results do not exclude the possibility of other pools, which may be small in magnitude, with turnover rates too rapid to be detected by the method of sampling via the milk fat, or a complex of pools with similar turnover rates. Indeed, many intermediate pools can be and have been postulated (5) for transport and recycling of fatty acid of both endogenous and exogenous origins through the intestinal wall, liver, adipose tissue, and finally to the mammary gland from whence the samples were taken. I t is

TABLE 5. Mean concentrations of plasma free f a t t y acids, plasma glucose, and serum heparinprecipitable lipoprotein total ester. Dieta~5, treatment a

No. of trials

FFA (t~m/liter plasma)

C HGRR LF HF

3 3 4 2

C vs. H G R R L F vs. H F

df 4 4

313 164 209 329

± 17.6 b ± 8.8 ± 19.5 ± 32.8

HPLP esters (/,,m/liter serum) 428 ± 28.2 767 ± 34.6 ND c ND

Glucose (mg/lO0 ml plasma) 70.6 -----2.38 77.5 ± 0.967 ND ND

Summary of test of significance P < 0.001 P < 0.01

P < 0.001

P < 0.01

a See Table ]. b Mean ± standard error. c Not determined. JOURNAL OF DAII~y SCIENCE VOL. 54, NO. 7

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P A L ~ Q U I S T AND CONRAD

unlikely that all adipose tissue pools have similar turnover rates. The turnover rates reported here would be an integral of the turnover rates of all adipose tissue pools since they would have been labeled with the 14C- palmitate dose in proportion to their turnover rates and would release the 14C fatty acid in proportion to their turnover rates. The observation of increased uptake o2 dietary fatty acids by adipose tissue with a fat-depressing dietary regime is in keeping with reports of generally increased metabolic activity of adipose tissue under these conditions (2, 4, 20, 28). I n addition to increased uptake of exogenous fatty acid, increased de novo synthesis of fatty acids by adipose tissue most certainly also occurs (2, 20, 28) which would further deprive the mammary gland of substrate for milk fat synthesis. The present study has broadened Van Soest's glueogenic concept (26) that high grainrestricted roughage diets may cause milk fat depression by reducing the amount of fatty acid mobilized from adipose tissue. I t appears that there is a "glucogenie response" which increases the competitive position of adipose tissue for metabolites required for milk fat synthesis. Even though milk fat palmitate arises nearly equally from plasma and de novo synthesis in the mammary gland (22), use of palmitie acid-l-14C in these experiments is justified on its wide occurrence in ruminant diets and ready availability in isotopically pure form at moderate cost. De novo synthesis in the mammary gland would not influence estimates of relative contributions of exogenous and endogenous fatty acid to plasma fatty acids but would have to be taken into account in calculation of transfer quotients of plasma fatty acids as pracursors of milk fat. This need not be a consideration in the present study which treats only the sources of milk fat which are derived from plasma. This study points up interesting areas for future research, namely, the turnover rates and relative uptake by the mammary gland of liversynthesized lipoproteins (LDL and VLDL) and ehylomicra synthesized by the gut wall. Additional studies on plasma free fatty acid turnover rates and of the inverse relationship between plasma free fatty acid and heparinprecipitable lipoprotein total esters between control and high grain-restricted roughage diets should prove valuable. That the high fat diet reduced by 25% the turnover time of the adipose pool may gain practical significance in instances where it beJOURNAL Ol~ DAIRY SCIEI~CE ~OL. 54, i~0. 7

comes important to decontaminate cows of pesticides or other undesirable fat-soluble residues. Consumption of both high- and lowfat diets and performance of the cows were equally acceptable during the relatively short terms of these trials. References

1960. Plasma nonesterified fatty acids in sheep. Australian, J. Agr. Res., 11: 58. Baldwin, R. L., H. J. Lin, W. Cheng, R. Cabrera, and M. Ronning. 1969. Enzyme and metabolite levels in mammary and abdominal adipose tissue of lactating dairy cows. J. Dairy Sci., 52: 183. Barry, J. M., W. Bartley, J. L. Linzell, and D. S. Robinson. 1963. The uptake from the blood of triglyceride fatty acids of cbylomicra and low-denslty lipoproteins by the mammary gland of the goat. Biochem. J., 89: 6. Benson, J. D., E. W. Askew, R. S. Emery, and J. W. Thomas. 1969. Effects of a restricted roughage ration on lipoprotein lipase and glyceride synthetase activities in bovine mammary and adipose tissues. Federation Proc., 28: 623. Emery, R. S. 1968. Personal communication. Flexner, L. B., D. B. Cowie, and G. J. Vosburgh. 1 9 4 8 . Studies on capillary permeability with tracer substances. In Cold Spring Harbor Symposia on Quantitative Biology. Biological Applications of Tracer Elements, 13: 88. Foleh, J., M. Lees, and G. H. Sloan Stanley. 1957. A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem., 226: 497. Garton, G. A. 1963. The composition and biosynthesis of milk lipids. J. Lipid Res., 4 : 237. Gellhorn, A., M. Merrill, and R. M. Rankin. 1944. The rate of transcapillary exchange of sodium in normal and shocked dogs. Amer. J. Physiol., 142: 407. Glaseoek, R. F., W. G. Duneombe, and L. It. Reinus. 1956. Studies on the origin of milk fat. 2. The secretion of dietary long-chain fatty acids in milk fat by ruminants. Biochem. J., 62: 535. Glascoek, R. F., V. A. Welch, C. Bishop, T. Davies, E. W. Wright, and R. C. Noble. 1966. An investigation of serum lipoproteins and of their contribution to milk fat in the dairy cow. Biochem. J., 98 : 149. Goodman, DeW. S., and R. P. Noble. 1968. Turnover of plasma cholesterol in man. J. Clin. Invest., 47: 231. Griel, L. C., Jr., and R. D. McCarthy. 1969. Blood serum lipoproteins. A review. J. Dairy Sci., 52: 1233.

(1) Annisen, E. F.

(2)

(3)

(4)

(5) (6)

(7)

(8) (9)

(10)

(11)

(12) (i3)

ORIGIN

OF PLASMA

(14) Gurpide, E., J. Mann, and E. Sandberg. 1964. :Determination of kinetic parameters in a two-pool system by administration of one or more tracers. Biochemistry, 3: 1250. (15) Hartmann, P. E., J. G. Harris, and A. K. Lascelles. 1965. The effect of oil-feeding and st~Lrvation on the composition and output of lipid in thoracic duct lymph in the lactating cow. Australian J. Biol. Sci., 19 : 635. (16) Harvey, W. R. 1960. Least-squares analysis of data with unequal subclass numbers. ARS~20-80. ARS, USDA. (17) Huber, J. T., R. S. Emery, 5. W. Thomas, and 1. M. Yousef. 1969. Milk f a t synthesis on restricted roughage rations containing whey, sodium bicarbonate, and magnesium oxide. 5. Dairy Sci., 52:54. (18) Lascelles, A. K., D. C. Hardwick, J. L. Linzell, and T. B. Mepham. 1964. The transfer of (3H) stearic acid from chylomicra to milk fat in the goat. Bioehem. J., 92: 36. (19) Nestel, P. J., H. M. Whyte, and DeW. S. Goodman. 1969. Distribution and turnover of cholesterol in humans. 5. Clin. Invest., 48 : 982. (20) Opstvedt, J., R. L. Baldwin, and M. Ronning. 1967. Effect of diet upon activities of several enzymes in abdominal adipose and mammary tissues in the lactating dairy cow. g. Dairy Sci., 50: 108. (21) Opstvedt, J., and M. Ronning. 1967. Effect upon lipid metabolism of feeding alfalfa hay or concentrate ad libitum as the sole

FATTY

(22)

(23)

(24)

(25)

(26)

(27)

(28)

ACIDS

1033

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