kinetics of triglyceride turnover of very low density lipoproteins of human plasma

Journal of Clinical InvestigationVol. 44, No. 11, 1965

Kinetics of Triglyceride Turnover of Very Low DensityLipoproteins of Human Plasma *

G. M. REAVENt D. B. HILL,4 R. C. GROSS,§ AND J. W. FARQUHAR WITH THETECHNICAL ASSISTANCE OF E. P. BROWN

(From the Department of Medicine, Stanford University School of Medicine, Palo Alto, Calif.,and the Department of Statistics, Stanford University, Stanford, Calif.)

Ahrens and co-workers (1) have collated andextended prior observations (2-7) that high car-bohydrate, low fat diets may cause hypertrigly-ceridemia in certain subjects. They termed thisphenomenon "carbohydrate-induced lipemia" (1).Despite uncertainties concerning the degree orduration of plasma triglyceride rise needed tomerit this appellation, this general response to ahigh carbohydrate diet is frequently observedin patients with evidence for accelerated athero-genesis (1, 8-10). Furthermore, elevated plasmatriglycerides are common in patients with "pre-mature" arteriosclerotic heart disease on ad libi-tum diets (11-16). Consequently, it becomes im-portant to define the factors controlling plasmatriglyceride concentration in man.

It can be assumed that changes in concentra-tion of water soluble macromolecules confinedto the plasma space, such as lipoproteins, resultfrom variations in rates of entry into and re-moval from plasma, or both. Hypertriglyceri-demia in patients with "fat-induced lipemia,"another and a relatively uncommon syndrome (1,

* Submitted for publication May 26, 1965, acceptedJuly 20, 1965.

Presented in part at the joint national meeting of theAmerican Federation for Clinical Research and theAmerican Society for Clinical Investigation, AtlanticCity, N. J., May 1964.Supported in part by U. S. Public Health Service re-

search grants AM 05972 and HE 08506 from the NationalInstitutes of Health, K3-HE 6003 from the Career De-velopment Review Branch, and FR-70 from the GeneralClinical Research Centers Branch, Division of ResearchFacilities and Resources.t Address requests for reprints to Dr. Gerald M.

Reaven, Dept. of Medicine, Stanford University Schoolof Medicine, Palo Alto, Calif. 94304.

4: Postdoctoral fellow in biostatistics, U. S. PublicHealth Service.

§ Postdoctoral research fellow, U. S. Public HealthService.

17), is associated with decreases in plasma post-heparin lipolytic activity (17, 18). This latterfinding suggests that the primary cause of thehypertriglyceridemia in fat-induced lipemia is de-fective removal of newly absorbed dietary chylo-microns due to deficiency in lipoprotein lipase orother tissue lipases. Although normal plasmapostheparin lipolytic activity has been described inpatients with carbohydrate-induced hypertrigly-ceridemia (1, 17), it does not necessarily followthat the plasma lipid change must be due toendogenous overproduction. Indeed, it has re-cently been suggested that the cause of hyper-triglyceridemia in this syndrome is due to a de-fect in removal of endogenous plasma triglyceride(8).To determine if a rise in plasma lipoprotein

concentration is caused primarily by increasedproduction or by decreased removal, it is neces-sary to compare turnover rate with concentrationthrough a wide range of these variables in adefined human population. At present, we findno data in normal or abnormal populations thatpermit such an analysis. However, we have re-cently devised and validated a model for trigly-ceride turnover in man using isotopically labeledglycerol as a precursor for plasma triglyceride(19). We report here our use of these techniquesin a study of the relationship between concentra-tion and turnover rate of plasma triglyceride ina group of human subjects. The results demon-strate a close dependency of these two variablesunder a wide variety of experimental conditions.

Methods

Subj ects selected for study were either assumed to haveno abnormality of lipid metabolism or were expected tohave wide variations of triglyceride concentrations in re-sponse to weeks of ingestion of either high or low fatdiets. Relevant clinical features of the 12 patients are

1826

LIPOPROTEIN TRIGLYCERIDE TURNOVER

TABLE I

General description of patients studied

Initials Age Sex Height Weight Clinical diagnoses*

cm kgG. B. 52 M 170 66.5 ASHDE. C. 41 M 164 89.8 ASHD, xanthoma tuberosum and planumF. C. 71 M 165 60.0 Carcinoma of lungP. G. 52 M 178 74.0 ASOC. H. 33 M 180 85.1 Subsiding jaundice secondary to

chlorpromazineR. L. 52 Xl 185 77.0 ASHDL. McD. 51 M 173 77.1 ASHDR. N. 44 M 170 73.2 ASHDJ. P. 48 M 174 74.5 ASHD, ASO, familial hypercholesterol-

emia, xanthoma tendinosumC. P. 38 M 188 103.1 ObesityG. S. 21 M 170 60.4 Bronchiectasis and membranous glomer-

ulonephritisG. T. 57 F 152 40.0 ASHD

* Description of terms: ASHD = arteriosclerotic heart disease; ASO = arteriosclerosis obliterans.

given in Table I. All but one (F.C.) were fully ambula-tory. Nine patients were fed isocaloric liquid formuladiets as the sole source of calories for 3 to 8 weeks beforemeasurement of triglyceride turnover. Techniques offormula feeding have been previously described (20);compositions as percentage of daily calories from pro-teins, fats, and carbohydrates were as follows: high fat,14: 69: 17, high carbohydrate, 14: < 1: 85. Formula in-gredients were derived from corn oil, dried skimmed milkpowder, and a partially polymerized dextrose product con-taining dextrins and small amounts of maltose.1 In threepatients the antecedent diet was an ad libitum, regular hos-pital diet. In six patients an attempt was made to producevariations in triglyceride concentration by dietary means,and triglyceride turnover was measured during both a highfat and a high carbohydrate dietary period. In three pa-tients, 10 additional studies were done on diets either highor low in fat, both with and without the daily administra-tion of 125 mg of chlorpropamide. Body weights of sub-jects were stable for at least 1 week before each study.Experimental techniques. Details of experimental meth-

ods were previously described (19) and with one excep-tion are unchanged. A general description of the stud-ies follows: Isotopically labeled' glycerol was given in-travenously after an overnight fast. Fasting was con-tinued and blood was removed at frequent intervals dur-ing the subsequent 8 hours. This blood was used to ob-tain Sf > 20 lipoproteins by ultracentrifugation at 140 C.A more complete and less variable recovery of Sf > 20triglyceride was obtained by use of the following modifi-cation of the technique previously reported (19): Poly-carbonate tubes 2 of 11-ml capacity wvere filled with only7 ml of plasma. Sf > 20 lipoproteins were removed byaspiration of the top 3 ml after ultracentrifugation (40 ro-tor, Spinco model L) 3 for 22 hours. Lipids were then

1 Dexin, Burroughs, Wellcome and Co., Tuckahoe, N. Y.2 "Autoclear" tubes and sealing caps, International

Equipment Co., Needham Heights, Mass.3 Beckman Instruments, Palo Alto, Calif.

extracted from these lipoproteins (19), and the triglycer-ides were isolated by thin layer chromatography, theirconcentration and radioactivity were determined, andspecific activities were calculated.

Calculations. A previous report from this laboratoryhas validated a two-compartment, nonrecycling model ofhepatic and plasma Sf > 20 triglyceride turnover in man(19). Turnover rates are calculated from the slope ofthe specific activity disappearance curve of endogenouslylabeled Sf > 20 plasma triglyceride after the intravenousinjection of isotopically labeled glycerol. The validity ofthis calculation is based upon our previous discovery ofthe rate-determining role in the two pool system ofplasma Sf > 20 triglyceride fractional turnover rates(19).The calculations are as follows: 1) Plasma volume is esti-

mated as 4.5%o of body weight (21). 2) Pool size =(milligrams Sf > 20 triglyceride per milliliter plasma) X(plasma volume in milliliters). 3) t- = half-time of dis-appearance of Sf > 20 triglyceride from plasma. 4)Fractional turnover rate = 0.693 . ti. 5) Turnover rate= (fractional turnover rate in hour-') X (pool size inmilligrams) + kilogram body weight.These studies were performed while plasma triglyceride

concentrations were constant. Therefore, turnover rateof plasma triglyceride is equal to plasma triglyceride fluxfrom liver into plasma. These data then allow one to de-fine the mathematical relationship of plasma triglycerideconcentration and turnover rate that exists in the popu-lation under study.

Results

The relationship between plasma Sf > 20 tri-glyceride concentration and turnover rate for eachpatient studied is tabulated in Table II, andportrayed in Figure 1. It can be seen that plasmatriglyceride concentration in nine to eleven de-

1827

REAVEN, HILL, GROSS, AND FARQUHAR

TABLE II

Relationship between plasma Sf > 20 triglyceride concentration and turnover rate

PlasmaCondition* volumet

ml

Pool size§

g

3.11

14.83

19.110.62

14.5252.474.31

20.0324.502.26

1.745.41

4.65

3.6219.83

2.723.12

2.354.73

2.2816.8330.340.63

1.102.16

tjhours

2.85.0

8.43.76.6

20.63.08.59.2

1.8

kjj

hours-i

0.2480.139

0.0830.1870.1050.0340.2310.0810.0750.385

1.9 0.3652.7 0.257

3.9 0.1783.4 0.2048.6 0.0812.7 0.2572.7 0.257

3.7 0.1875.8 0.119

3.7 0.1874.1 0.1698.3 0.0831.3 0.5331.7 0.4082.4 0.289

* Conditions: HF = high fat diet; HC = high carbohydrate diet; chlor. = chlorpropamide; ad lib. = ad libitum diet.t Plasma volume is assumed as 4.5% of body weight.CV = Coefficient of variation = (standard deviation - mean) 100. Values for the mean were obtained from

nine to eleven plasma samples drawn during 6 to 8 hours of the experiment.§ See Methods for techniques for calculation of pool size, fractional turnover rate (k), ti, and turnover rates.11 Fractional turnover rate; see Methods for technique of calculation.

terminations was relatively constant during the6 to 8 hours that triglyceride turnover rate was

determined (Table II). The results indicatethat at each higher plasma triglyceride concentra-tion there were generally associated increases intriglyceride turnover rate. This relationship be-tween plasma triglyceride concentration and turn-over rate was analyzed by Spearman's rank cor-

relation coefficient (22), and the hypothesis thatthese two variables are unrelated can be rejectedat the level of less than 0.0001.

It can also be seen that concentration andturnover rate were not linearly related through-out, and that concentration rises steeply after

turnover rates begin to exceed 15 mg Sf > 20triglyceride per kg body weight per hour.The shape of the curve suggests that the re-

moval mechanisms for plasma Sf > 20 trigly-cerides follow the kinetics of a saturable system,and that the plot is not compatible with simplediffusion as the mechanism of transport out ofthe system (23). To more precisely define theserelationships, a mathematical model was formu-lated based on the following assumptions: 1) Asteady rate of hepatic secretion of Sf > 20 lipo-proteins maintains a steady concentration ofplasma Sf > 20 triglycerides. 2) There is some

large number of "removal sites" through which

Sf >20 TG

Patient

G. B.E. C.

F. C.P. G.

C. H.R. L.

L. McD.

R. N.

J. P.

C. P.

G. S.G. T.

HCHCHC +

chlor.Ad lib.HFHCAd lib.HCHCHFHF +

chlor.HCHC +

chlor.HFHCHFHFHF +

chlor.HCHC +

chlor.HFHCAd lib.

HFHC

2,9934,041

4,0412,700

3,3003,3003,8203,4653,4653,470

3,4703,470

3,4703,2943,2943,3523,352

3,3523,352

3,3524,9504,9502,718

1,8001,800

Mean CVt

mg per 100 ml

104±- 5

367±- 7

473 i 4

23 4- 17

441 i 121,590± 9

112 4i 8

57870765 ±- 25

50 ±t 30156 ±t 23

134 - 10

110 10602 ±t 9

81 ±- 2693 ±- 17

70 - 13141 ±t 25

68 ±- 40

340 11613 - 13

23 - 1761 ±- 43120 ±t 8

Turnoverrate

Sf > 20 TG

mg per kgper hour11.723.0

17.72.0

20.623.810.021.024.911.3

8.318.1

10.810.022.09.410.7

5.97.6

5.824.822.95.6

11.315.5

1828

LIPOPROTEIN TRIGLYCERIDE TURNOVER

H1

F-00

:

sal

FIG. 1. RELATIONSHIP BETWEEN St > 20 TRIGLYCERIDE TURNOVER RATE (V)AND CONCENTRATION [S]. These values are plotted from each of the 25studies listed in Table II. Units: V (velocity) = Sf > 20 triglyceride turn-

over per kilogram body weight per hour; [S] = milligrams Sf >20 tri-glyceride per 100 ml plasma; Vmax =maximal estimated velocity; Km= [S]at i maximal velocity.

Sf > 20 triglycerides may leave the plasma. 3)If a removal site is "occupied," it is unavailablefor use by another triglyceride until the "removalprocess" has been completed. 4) The rate oftotal removal of triglyceride from the system isdirectly proportional to the number of removalsites occupied. 5) The rate at which triglyceridesarrive at the removal sites is directly proportionalto the plasma triglyceride concentration.Terms and formulas to express these assump-

tions in the kinetics of a saturable system follow.Because of its common use, terminology analo-gous to the Michaelis-Menten formulation (24)was used. [S] = concentration of Sf > 20 plasmatriglycerides in milligrams per 100 ml = concen-

tration of substrate. CO = concentration of re-

moval sites in sites per 100 ml plasma. k =fractional turnover rate. CS = concentration ofoccupied removal sites in sites per 100 ml plasma.V = input rate of Sf > 20 triglycerides from liverto plasma = removal rate of Sf > 20 triglyceridesfrom plasma = velocity of the removal reaction= milligrams Sf > 20 triglycerides per 100 mlplasma per hour. P = Sf> 20 triglycerides re-

moved from plasma = products.

Then,ki k3

Co+[S]< 'zCS- ,Co+P [1]

k2

Then, the equations governing this system are:

d[S]/dt= -k1[S]j(CO-CS)+k2 CS+V. [2]

dCS/dt = kl[S] (CO- CS) - (k2+k3) CS. [3]

And, in the steady state condition, one has

d[S]/dt =dCS/dt = 0.

Solving these equations for V, we obtain:

V = K[ ]+1Swhere Vmax = k3C.,ndESk k

andKm =k+k

[4]

ES]

(Vmax = maximal turnover rate allowed in thissystem, and Km = substrate concentration, [S],at i Vmax.)

Figure 1 illustrates the results of these experi-ments when [S] (concentration of plasma Sf> 20 triglycerides) is plotted against V (turn-over rate). The turnover rates of Figures 1 and2 are calculated per unit body weight rather than

v

1829

REAVEN, HILL, GROSS, AND FARQUHAR

200 400 600. 80S 000 120 K400 600

[SI

FIG. 2. LINEAR TRANSFORM OF THE DATA OF FIGURE 1

(1/[S] AGAINST V) TO ACHIEVE A MORE ACCURATE ESTI-

MATE OF Km AND VmaX THAN IS POSSIBLE FROM FIGURE 1.Slope obtained as the best fit line through the data points.V.ax = 1/slope, and Kma. = (intercept of slope with ordi-nate) (Vmax).

unit plasma volume (see Equation of calcula-tions.) As Vma, is approached, the rate of re-

moval appears to be independent of substrate con-

centration, and the reaction approaches zero orderkinetics. At lower levels of substrate concentra-tion, removal rate appears to be directly propor-

tional to substrate concentration, and the relation-ship seems to be first order.To calculate Vma. and Km more accurately than

possible from the curve of Figure 1, a linear trans-form of the Michaelis-Menten formulation of1/[S] against V was constructed (Figure 2).The slope of the best fit line through these data(Figure 2) was 0.0095. Since in this type ofplot, Vma, = 1/slope (24), then Vmax = 26.5 mg

Sf > 20 triglyceride per kg body weight perhour. Also in this system, Km equals the prod-uct of the intercept on the vertical axis timesVmax. Therefore, Km = 118 mg Sf > 20 trigly-ceride per 100 ml plasma.

Five paired studies were designed to investi-gate the effect of chlorpropamide on triglycerideturnover. Results showed that turnover rate,when measured after several weeks of chlorpropa-

mide treatment, was always lower than in pre-vious studies on the same diet (Table II). Itwas not possible to discern any systematic de-viation of these data from the general shape ofthe curve of Figure 1.

Also, there were six comparisons of high andlow fat diets (Table II). In one subject (J.P.),little difference in Sf > 20 triglyceride concentra-tion or turnover was noted. In the remainingfive comparisons, lower concentrations werepresent in the high fat diet; all pairs but one(C.P.) resulted in distinctly lower turnover rateson the high fat diet.

Discussion

The concentration of any solute in plasma willtend to rise and fall as its flux in or out ofplasma is varied. A rise in plasma concentrationto a new steady state level can only result fromeither increased entry rate or decreased removalrate of the solute in question. During nonsteadystate conditions, as the solute concentration isrising, its removal rate from the plasma mustbe less than its entry rate. Eventually, effluxwill again equal influx, and solute concentrationwill now be stable at its higher plasma level.Consequently, whenever solute concentration hasincreased, removal mechanisms have failed tomaintain homeostasis. However, it does not fol-low that a primary defect in solute removal isresponsible for the rise in concentration. Toestablish the presence of such removal defects itis necessary to find a plasma concentration ofSf > 20 triglyceride associated with a lower turn-over rate than is normally present for that con-centration. Our results (Figures 1 and 2) argueagainst the existence of primary removal defectsbecause they deny the presence of two or moredistinct population groups differing only in plasmatriglyceride removal rate. In fact, the homogene-ity of the fit of the experimental data to a singlecurve (Figures 1 and 2) indicates that the popu-lation studied was remarkably homogeneous intriglyceride removal efficiency. In these studies,higher plasma Sf > 20 triglycerides were gen-erally accompanied by higher turnover rates ofthis substance (Table II, Figure 1). At eachincrement in plasma concentration more triglycer-ides were being made and secreted into plasma

1830

LIPOPROTEIN TRIGLYCERIDE TURNOVER

and more triglycerides were being removed fromplasma. Production and removal were equalsince each experimental point represented a steadystate in concentration. Higher plasma concen-tration occurred because removal mechanisms didnot keep pace with increased production, but in-creased production was the primary cause for therise in plasma Sf > 20 triglyceride concentration.

This does not mean that certain individualswill not demonstrate important variations fromthe relationship that has been described, and inthese instances hypertriglyceridemia may well re-sult from primary removal defects. For example,it seems quite likely that patients with "fat-in-duced hypertriglyceridemia" would demonstratean elevated level of plasma triglyceride in asso-ciation with low turnover rates. Furthermore,the recent report of decreased plasma postheparinlipolytic activity in patients with myxedema (25)suggests that hypertriglyceridemia in this dis-order may also result from lessened removal ef-ficiency. However, in contrast to these and otherunusual disorders, the patients we studied shouldbe more typical of the general population. Allof our subjects were consuming constant diets,their body weights were stable, and individualswith fasting hyperglycemia or with fat-inducedhypertriglyceridemia were excluded. Within thisgroup of patients varying from clearly normal toabnormal in plasma triglyceride concentration,turnover rates were determined at different tri-glyceride levels that were varied by dietary altera-tions or by administration of chlorpropamide.By these means some of the group were raisedor lowered in Sf > 20 triglyceride concentrationand in turnover rate to levels that bracketed thetriglyceride concentrations of others of the group.This interlocking of responses through the widerange of observed concentrations suggests thata larger sample of the general population wouldfollow the observed relationship between plasmatriglyceride concentration and turnover rate (Fig-ures 1 and 2).The data relating plasma triglyceride concen-

tration to turnover rate were presented in thenomenclature of the Michaelis-Menten formula-tion, not because the precise enzymatic nature ofclearing has been identified, but because the ex-perimental data are consistent with such kinetics.The function relating concentration to turnover

rate in these studies is clearly explicable by asaturable system, perhaps an enzyme dependentmechanism, and cannot, by its nonlinearity, beattributed to simple diffusion. Moreover, thereis much evidence in numerous mammalian speciesthat an enzymatic process is responsible for clear-ing triglyceride of either chylomicrons (26) or oflow density lipoproteins (27). Although otherenzymes have not been excluded (17), the majorenzyme in this reaction is very likely lipoproteinlipase, an enzyme first isolated and characterizedby Korn (28). This enzyme is found in manytissues, but it is in highest concentration in adiposetissue (29, 30) or heart muscle (29) of manyanimal species. Although the precise anatomicsite of clearing is unknown, Robinson and Harrishave presented evidence for an endothelial loca-tion of lipoprotein lipase (31). This suggests anaction within capillary beds of the principalconsumers of plasma triglyceride, striated muscleand adipose tissue.

Regardless of the exact nature of the enzymesconcerned with removal from plasma of the lipidmoieties of low density lipoproteins, it seems clearthat knowledge of turnover rates is essential inorder to understand the relationship betweenhepatic triglyceride secretion and abnormalities oflipid metabolism in man. Our data (Figures 1and 2) clearly show that sole reliance upon changein concentration may mislead one in attempts torelate these changes with the more importantissue of how much triglyceride is being made andsecreted by the liver. For example, an increasein secretion (turnover) rate of from 10 to 15mg triglyceride per hour per kg body weightis associated with a rise of only 40 mg Sf > 20triglyceride per 100 ml plasma. In contrast, asimilar increment in turnover from 20 to 25 mgtriglyceride per hour per kg body weight wouldresult in an increase of 600 mg Sf > 20 triglycer-ide per 100 ml plasma. However, it should benoted that this degree of "saturability" may notoccur in all species. Our studies in the dog, forexample, indicate that Sf > 20 triglyceride con-centration and turnover rate are more linearlyrelated and that dog turnover rates exceed three-fold those of man at the Km of man (32). Theserelationships, considering the low Km of man(118 mg Sf > 20 triglyceride per 100 ml plasma),allow one to predict greater lipemia in man than

1831

REAVEN, HILL, GROSS, AND FARQUHAR

in the dog for comparable stimulation of hepatictriglyceride synthesis.

Finally, our observation that chlorpropamidedecreased Sf> 20 triglyceride turnover rates inthe three subjects studied is intriguing in viewof recent observations that patients with eithercarbohydrate-induced hypertriglyceridemia (8-10,33) or "essential" hypertriglyceridemia (34) ap-pear to have a very mild form of maturity onsetdiabetes. Studies are in progress to confirm thiseffect of chlorpropamide and to relate it to ourcurrent hypothesis that plasma insulin levels arean important determinant of the rate of hepatictriglyceride synthesis and secretion that can beinduced in man by high carbohydrate diets (10).

Summary

These studies establish the relationship betweenthe concentration and turnover rate of plasmaSf > 20 triglyceride in a defined human popula-tion. The results demonstrate that triglycerideconcentration and turnover rate are highly cor-related in this group, and that higher concentra-tions are associated with higher turnover rates.Furthermore, the function relating these two vari-ables is consistent with a saturable, and possiblyenzyme dependent, mechanism for removal ofthis lipoprotein triglyceride from plasma. Sincethe clearing efficiency within this group seemsremarkably homogeneous, increased productionrather than a triglyceride removal defect is there-fore responsible for rising plasma Sf > 20 tri-glyceride concentration. Finally, the low sub-strate concentration at 4 maximal turnover rateof this system (118 mg Sf > 20 triglycerideper 100 ml plasma) attests to the ready satura-bility of triglyceride removal mechanisms in man.

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