Europ. J. clin. Invest. 4, 109-114 (1974)

Comparison between Fractional Turnover Rate

(Intravenous Fat Tolerance Test) in Man* of Endogenous Plasma Triglycerides and of IntralipidQ

S. Rijssner, J. Boberg, L. A. Carlson, Ulla Freyschuss, and B. W. Lassers King Gustaf V Research Institute and the Departments of Internal Medicine and Clinical Physiology,

Karo~inska Hospital, Stockholm and the Department of Geriatrics, University of Uppsala, Uppsala, Sweden

Received: June 12, 1973, and in revised form: December 10. 1973

Abstract. The validity of the intravenous fat tolerance test (NFTT) as a tracer for the fractional turnover rate of endogenous plasma triglycerides (TG) has been studied in 32 fasting men with either normal or elevated plaama TG con- centrations. The endogenous plasma TG turnover was deter- mined by sampling arterial and hepatic vein blood, deter- mination of splanchnic net secretion of plasma TG and calcu- lation of fractional TG turnover rate. h t e r the fractional elimination rate of exogenous TG was determined following a single IV injection of IntralipidQ (IVFIT). The TG frnctiond removal rate conatants derived from them two tests were significnntly correlated (r = 0.6-0.7) but IVFTT gave higher

absolute values. A statistical evaluation showed that the error for the determination of the fractional turnover rate of endo- genous TG and the IVFTT were about 30-50% and 10% respectively. It is probable that the correlation would have been still better if the error for the estimation of endogenous plnsma TG turnover had been lower. A significant negative correlation was found between very low density lipoprotein-TG concentration and fractional removal rate of both endogenous and exogenous plasma TG (r = - 0.7--0.8).

Key WOrJs: Human, intrnlipid", intravenous fat tolerance test, plasma triglyceride, plasma triglyceride fractional turnover rate.

Hypertriglyceridaemia (HTG) is a common meta- bolic disorder in man, and is frequently seen in patients with clinical manifestations of atherosclerosis [ l , 21. Fasting HTG is often asymptomatic [3] and is an important "risk factor" for future development of coronary heart disease [4].

HTG may result either from an increased secretion (turnover rate) of triglycerides (TG) into plasma or from an impaired removal (fractional turnover rate) of TG from plasma. In recent years a number of in- vestigations of plasma TG transport have been made using widely different techniques ([5, 61 cf. [7], cf. [8, 91). The most direct method has been the measure- ment of net splanchnic production of plasma TG [9]. These studies have indicated that there is a wide range of the values for fractional turnover rate of plasma TG in HTG and that an impaired removal rather than increased turnover of plasma TG is the most common cause of HTG in man [5, 6, 10-133. The fact that there is a wide range of the fractional turnover rates among subjects with similar plasma TG concentration makes it important to determine not only plasma TG concentration but also plasma TG fractional turnover rate. It is thus important to have an estimate of plasma TG fractional turnover rate to elucidate pathogenesis of HTG and mode of action of hypolipidaemic drugs as well BS to aid in clsssification of HTG. However none of the available methods for estimation of plasma TG turnover is simple enough to be used in clinicaI routine. In an attempt to find a simple method for estimation of plasma TG fractional * Supported by granta from the Swedish Medical Remarch Council (19s-204, 19P-2628) and Vitrum AB, Stockholm, Sweden.

turnover rate we have developed an intravenous fat tolerance test (IVFTT) using the fat emulsion Intra- lipid@ [14, 151. The kinetics for the removal of Intra- lipid8 from blood are identical to those for chylo- microns [16]. Furthermore when IntralipidO and chylomicrons are incubated in vitro with post heparin plasma the lipoprotein lipase enzyme kinetics are also almost identical for the two substrates [17]. Since removal of endogenous plasma TG appears to occur by the same mechanisms as for chylomicrons we have suggested that the IVFTT may be used as a method to study the removal of endogenous plasma TG [15].

The present series of investigations wm under- taken to compare the fractional turnover rates of endogenous plasma TG obtained by studies of the splanchnic TG secretion {9] with the results of the IVFI'T in healthy control subjects and patients with HTG.

Methods Sub jeck

Thirty-two healthy male blood donors or patients with hyperlipoproteinaemia between 26 and 62 years of age volunteered to take part in the study. Each investigation began a t 8 a.m. The subjects had fasted overnight and refrained from smoking, but were not given instructions of any special diet prior to the investigation. All patients had been found to have a fasting plasma TG concentration of more than 2mmol/l on at least one occasion before the study. No patients or control subjects had clinical diabetes w judged by the absence of glucosuria and a fasting blood sugar concentration below 105 mg/100 d. No other endocrine disorders or acute diseases were

110 S. Rassner et nl.: Plasma TC. Turnover and IVFm

INTRAENK‘S FAT TOLERANCE SllM Fig. 1. Graph showin each subject’s TG concentrations at the two studies: ( I ) T$FIT study (abcissa) and (2). Fractioiial endogenous plasma TG turnover rate study (ordinate). The

equations y = x, y =q z and y =& z are also indicakl

present as judged from the histories and physical examinations. The subjects were divided into two groups : “normals ” or “ hypertriglyceridaemics ” on the basis of their values for plasma TG concentration compared with the Stockholm population ( 1 8).

Design of the Study The investigation of endogenous plasma TG turn-

over was followed by t,he IVFTT within one month in 20 of the 32 subjects and within six months in the others. The methods for determination of endogenous plasma TG turnover as well as the values obtained for endogenous plasma TG fractional turnover rate have been described previously in detail [9, 61. In principle one catheter was inserted into a cubital vein for infusion of albumin bound tritiated palmitate and lndocyanine green. For blood sampling a second catheter was inserted into the hepatic vein and a third into a brachial artery. Blood samples were drawn simultaneously from the artery and the hepatic vein catheters a t 180, 210 and 240 minutes after the start of the tritiated palmitate infusion. Concentrations of plasma FFA, TG, glycerol and Indocyanine green and the radioactivity of plasma FFA and TG were determined in these samples. A t the beginning of the study blood was drawn for determination of the TG content in plasma very low density lipoproteins (VLDL) separated by ultracentrifugation a t d = 1.006. A t the end of the study the plasma volume was measured.

In the I V F l T [15] catheters were inserted into a cubital vein of ewh arm. One was used for the in- jection of 0.1 g TG/kg body weight of 20% Intra- lipid@ (kindly supplied by AB Vitrum, Stockholm) and the other for blood sampling. Blood for deter-

mination of plasma TG and cholesterol concentrations and a blank for the nepheiometric determinations was taken a t zero time and then IntralipidB was given as a bolus injection as fast as possible, generally within one minute.

3 ml of blood were then taken every five minutes into heparinized tubes for forty minutes and the light scattering index of plasma samples were determined by nephelometry [15].

Calculations The fractional turnover rate for plasma VLDL-TG

was calculated by dividing the values for plasma TG turnover rate by the plasma VLDL-TG pool. The TG turnover rate was calculated either as net splanch- nic secretion rate (hepatic venous-arterial difference of plasma TG concentration times estimated splanchnic blood plasma flow) or as blood p l a s m clearance m t e . The latter was determined in the following way. From the splanchnic secretion of labelled TG into the blood stream over a certain period of time the in- crease of the labelled blood plasma TG pool over the same period was subtracted. This difference was the amount of labelled plasma TG which had been removed from the circulation. To obtain the cor- responding chemical amount being removed per minute the difference was divided by the time period and the mean specific activity of the VLDL plasma TG of the same period.

The value of “fractional turnover rate” of endo- genous plasma TG was expressed as per cent of the pool turning over per minute.

A semilogarithmic plot of the light scattering indices against time during the IVFTT was found to be linear in all cases. The slope of the line was calcu- lated by the method of least squares [19] and ex- pressed as k, per centlminute. This value was used as the fractional removal rate of IntralipidO. In order to evaluate the variations of a correlation analysis of endogenous fractional turnover rates and IVFTT, the errors of each method were calculated from the means of the standard errors of the mean of the hepatic venous-arterial differences of unlabelled or labelled plasma TG concentrations. The average of the standard errors of the mean thus obtained was expressed in per cent of the average hepatic vein- arterial differences for each subject. The error of the WFIT was calculated from duplicate tests.

Results Subjects

The fasting plasma TG concentrations of the sub- jects a t the two separate investigations are given in Fig. 1 and Table 1. Most subjects had similar plasma TG concentrations on the two different study oc- casions. However in seven subjects the ratio between the two values was outside the arbitrarily chosen interval 7/10 to 10/7 (see Fig. 1 and Table I ) . The

S. %saner et ul.: Plaema TG Turnover and IVFTT 111

Table 1. Characteristics of the subjecta having similar (group A) and dissimilar (group B) plaama TG levels on the two occaeiom (eee text). Mean values f SEM are shown. Rangee within brackets

Body weight‘ Weightmeight Plasma TG (mmol/l)

endogenous TQ exogenous removal - n Age

(Yea4 (kg) (indexb) removal study study (IVFTT)

Group A “ h’ormal subjects ” 13 39&4 73f 3 0.98 f 0.03 l.34f0.18 1.29f 0.17

Patients with 12 48&2 79f4 l.Mf 0.04 5.00,t 1.11 5.22f 1.06 (2-2) (59-87) (0.78 - 1.11) (0.53 - 2.37) (0.65 - 2.20)

“hyperlipoproteinan3emia” (35-61) (63-105) (0.93-1.35) (2.32 - 16.50) (2.51-15.37)

Group B B.L. 54 83 0.98 1.38 6.36 0s. 55 71 0.99 2.39 1.59 B.A. 53 94 1.34 1.76 0.94 H.M. 27 75 1.01 2.05 0.75 A.A. 43 80 1.03 3.62 2.45 K.K. 44 86 1.08 4.63 2.80 T. J. 52 70 1.02 8.92 13.73

a Values during the endogenous TG removal study. b weight in kg

height in cm-100

O

a

* * 0

a a

0

0

a

a

a

I I I I ob 1 b ,g *A m 6 n & d R A T E i s w n D ( I

I

Fig. 2. Relationship between the “fractional turnover rate” determined by the chemical plasma TG secretion method and the intravenous fat tolerance. Closed circlea repreeent normo- triglyceridnemic subjects, o n circles represent hypertri- glyceridaemic subjecta. All Eta from eubjecta in group A

(Table 1)

25 subjects having a ratio between 7/10 and 1017 were allocated to a group called A and the seven subjects falling outside to a group B. The characteristics of the subjects in group A and B are summarised in Table 1. The weights and the weightiheight indices given were obtained a t the first etudy. The weight change between the studies calculated 88 per cent without sign was 1.3 f 0.3% (SEM) in group A and 1.6&0.6% in group B. The lipoprotein patterns analysed by both ultracentrifugation and paper electrophoresis and classified accon3.q to the WHO classification [20] were done at the first study. 8 Europ. J. din. Invut.. VoL 4

*t

0 O O O

a

a

a

”* FRbcm(4L-RATE(cLEARAHI)

Fig. 3. Relationship between the “fractional turnover rate” determined by the pleema TG clearance method and the intravenous fat tolerance. Symbola and subjects as in Fig. 2

Group A consisted of 1 3 subjects with “normal” concentrations of cholesterol and serum TG and normal serum lipoprotein patterns and 12 patients with ‘ I hyperlipoproteinemia”. Of the patients with hyperlipoproteinaemia one had a type IIB, one a type III and 10 a type IV lipoprotein pattern.

In one subject an extra band between pre-8 and a waa found and in two subjects a “sinking pre-/? band” waa observed.

Group B consisted of three subjects with “normal” serum lipoproteii patterns and four with serum lipo- protein abnormalities (one typeIIB, two typeTII and one type IV pattern).

112 S. %saner ef al. : Plasma TG Turnover and IVFTL’

0

10.0

5.0

- . 2 E 2 1.0

!2 ’ 0.5 -I

0.1

0 0

0 0

0

0 0

0 0

0 .. ..

. ..

1.0 5.0 10.0 lnlravenous fat td~ance%/min

Fig. 4. Relationship between the intravenous fat tolerance and plasma VLD-TG concentration. Logarithmic scnles.

Symbols and subjects as in Fig. 2

0

10.01

5.01 o n

0

0

0

0 .. .*

. .

0.1 1 02 0.5 1.0 5.0 10.0

”Fractional turnowr rate: Chemical TG secretion %/min

Fig. 6. Relationship between the “fractional turnover rate” determined by the plasma TG secretion method and plaama VLD-TG concentration. Logarithmic ecales. Symbols and

subjects as in Fig. 2

1o.c

5.0

- h 0 E E

ll $2 1.0

2 0.5

0.

0

D 0

0 0

0

0 0 0

0 0

0 .

0.2 0s io 5.0 Fractional turnover rat$-Plasma

TG clearance%/min

Fig. 6. Relationship between the “fractional turnover rate” determined by the plasma TG clearance method and plasma VLD-TG concentration. Logaritlunic scales. Symbols and

subjects as in Fig. 2

Plasma TG Turnover Rates Plasma TG turnover rates were determined as

described previously [6]. In the 13 normotriglycerid- aemic subjects the mean plasma TG turnover rate as determined by the chemical TG secretion method was 20 f 3 (SEM), the range being 5-42 pnol/rnin/m2 body surface area. The corresponding figures for the 12 hypertriglyceridaemic subjects were 30 f 7, range 7-73 pmol/min/ma body surface area. This difference between the means was not significant.

Re?noval of P h TG The I V F ” was significantly correlated with the

fractional removal rate of endogenous plasma TG determined either by the splanchnic TG secretion method (Fig.2) or by the plasma TG clearance method (Fig. 3). The fractional removal rates of both endogenous and exogenous plasma TG were lower in subjects with hypertriglyceridaemia than in subjects with normal TG concentrations (Figs.2 and 3). Table 2 and Figs. 2 and 3 include only the values for those subjects belonging to group A. If the subjects

Table 2. Equations for the correlation curpea between “fractional removal rate” (2) determined by different methods or ITFIT imd W - T G concentration (y) (group A subjects, n = 25)

Method r-value Significance

Intravenous fat tolerance y= -1.48 clog ~+2.13 -0.75 p<O.Ool Chemical plasma TG secretion clog y = -0.85 elog ~+0.14 -0.78 p<O.Ool Plaama TG clearance clog y = -0.77 40g ~ f 0 . W -0.05 p<O.Ool

S. Rhner d d.: Plasma TG Turnover and IVFTT 113

Table3. Comparison between the endogenous plasma TG turnover rate determined by either the chemical TG secretion method or the plasma TG clearance method and the intravenous

fat tolerance in eubjecta of group A

Comparison z, = fractional z, =fractional turnover rate turnover rate (chemical (plasma clear- method) ance method)

fat tolerance y, = intravenous

(n = 25)

y, = y1

(n = 24)

Equation r-value

y, = 0.58 2,+3.37 y* = 0.91 2,+2.73 0.60 0.71

Significance p<O.Ol p <0.001 of r

5 1.68 1.70 2i 4.35 4.28 Mean deviation from regression sx sz z

SY % -

1.94 1.90

116

44

1.31 1.68

77

39

Ehrlier obtained analytical errors of the methods expressed in % J

€ 2 50 32 t Y 13 13

* The error of the I V F a waa calculated from earlier duplicate

determinations according to the formula g, n=91 [15].

The error of the fractional turnover rate of endogenous plasma TG was calculated from earlier studies 88 mean values of standard errors of the mean of the hepatic venous-arterial differences of unlobelled (n = 49) or labelled (n = 35) plasma TG concentrationa [el. The latter standard errors were average values for each subject.

with pronounced variation in plasma TG concentra- tion (group B) are included the correlations remain significant but the r-values are reduced from 0.60 to 0.46 and from 0.71 to 0.56 respectively.

There is a significant hyperbolic relationship between intravenous fat tolerance and concentration of serum VLDL-TG corresponding to a linear function in a double logarithmic plot (Fig. 4). Similar sigruficant relationships exist between “fractional turnover rate ” of endogenous plasma TG and the concentrations or serum VLDL-TG (Figs. 5 and 6). The equations for these relationships are summarized in Table 2.

Discussion In the present study the fractional turnover rate

of endogenous plasma TG measured by two direct methods both of which involve hepatic vein catheteri- aation has been compared in the same individual with the measurement of a fractional elimination rate of 8.

TG (NFTT) using an intravenous injection of an artificial lipid emulsion (IntralipidG3). The rate constants given by the different methods were signrficantly correlated and indicate that the IVFTT reflects endogenous TG fractional removal.

Since the investigations were performed on different days without control of environmental factors, i t is natural that different plasma TG concentrations were found in one and the same subject. It cannot be excluded that these changes in concentration had been caused by variations of the fractional removal rate.

For this reason the subjects were divided into two groups, group A with similar and group B with dissim- ilar plasma TG concentrations in the two studies. There was a better correlation between the endo- genous and exogenous removal rates in group A than in group B.

The correlation coefficients for the comparison between the fractional removal rates of endogenous and exogenous TG were in Group A 0.60 and 0.71 depending on which method was used for estimation of endogenous plasma TG: transport. Although the correlations are sigmficant Figs. 2 and 3 reveal that there is a considerable variation between exogenous and endogenous fractional turnover ratcs. We believe that this variation is in part caused by the great analytical error in the methods for estimation of endogenous TG turnover. A statistical analysis of the role of the analytical error for the variation between the methods is given in Table 3. The variations s, and + calculated from the regression analyses and expressed as mean deviation from regression in per cent of the means 2 and comprise both inter- and intraindividual variations. The interindividual varia- tion which is a part of s, and c may very well be great without affecting the r-value. However the intraindividual variation which also is characterized by the analytical errors (& and f,,) will be of import- ance for the correlation coefficients ( r ) . Thus greater analytical errors will cause lower r-values. This can be seen in Table3 where the correlation coefficient between z, and yl was 0.60 and between x2 and y2 was 0.71. The corresponding mean deviation from regression of 21 and x, were 116% and 77% and the corresponding analytical- errors 50% and 32%. Although there is a significant correlation between the fractional turnover rate of endogenous plasma TG and the fractional elimination of IntralipidOJ from the blood the latter is removed more rapidly. Several possible explanations exist for this difference. The methods of determining endogenous plasma TG turnover rate underestimate the fractional turnover rate since the splanchnic uptake of plasma TG is excluded because we have used hepatic venous- arterial and not hepatic venous-portal differences for plasma TG [6]. 20% IntralipidB which was used in th study has previously been shown to be removed at a higher rate than 10% Intralipid@ [15]. This

114 S. R68sner e i al. : P h m a TO Turnover and IVFTT

difference may be explained by differences in emulsion particle size. As the 20% IntralipidB haa larger emuision particles than ordinary chylomicra [21] thia variation in particle size may explain the difference in fractional turnover rate. Similarly larger chylo- micron particles are removed more rapidly than smaller particles [22].

The significant correlations between fractional turnover rates of exogenous and endogenous plasma TG found in the present study make i t likely that such correlations may be found in larger samples with a wide spectrum of disorders causing endogenous HTG.

In the clinical investigation of plasma TG meta- bolism the IVFTT can be applied as an additional tool measuring the mechanisms involved in plasma TG removal.

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Dr. S. Rijssner King Guetaf V Research Inatitute s-10401 Stockholm 60 Sweden