Clinical Science and Molecular Medicine (1973) 45, 313-329.
T H E L I P I D S A N D LIPOPROTEINS O F H U M A N
P E R I P H E R A L L Y M P H , WITH OBSERVATIONS O N T H E
T R A N S P O R T O F CHOLESTEROL F R O M P L A S M A A N D
TISSUES INTO LYMPH
D. R E I C H L , L. A. SIMONS, N . B. MYANT, J. J. PFLUG A N D
G. L. MILLS
Medical Research Council Lipid Metabolism Unit, Hammersmith Hospital, London,
Department of Experimental and Reconstructive Surgery, Royal Postgraduate Medical School
and Hammersmith Hospital, London, and Courtauld Institute of Biochemistry,
Middlesex Hospital Medical School, London
(Received 2 April 1973)
SUMMARY
1. The lipids and lipoproteins of lymph obtained from the dorsum of the foot were
examined in seven human subjects.
2. The concentration of total cholesterol in lymph was about one-tenth that in
plasma and was significantly correlated with the plasma total cholesterol concentration. The ratio of esterified to total cholesterol in lymph was similar to that in plasma.
3. Triglyceride was detectable in lymph, but the concentration was less than
one-tenth that in plasma and was unrelated to the plasma triglyceride concentration.
4. No lipase activity was detectable in lymph, either before or after intravenous
injection of heparin.
5. Cholesterol-esterifying activity was detected in four samples of lymph.
6. The major lipoprotein antigens of human plasma (apo-A, apo-B and apo-C)
were present in whole lymph, but their distribution in fractions of different density
was different from that in plasma.
7. ['4C]Cholesterol, injected intravenously, appeared in lymph within 30 min of the
injection, indicating that some of the cholesterol in lymph is derived directly from
plasma.
8. At intervals greater than 29 days after a single intravenous injection of [14C]cholesterol, the specific radioactivity of lymph cholesterol was greater than that of
plasma cholesterol, indicating that some of the cholesterol in lymph is derived from
tissue pools of cholesterol with slow turnover.
Key words : lymph lipoproteins, lymph lipids, extravascular transport of cholesterol.
Correspondence : Dr D. Reichl, Medical Research Council, Lipid Metabolism Unit, Hammersmith
Hospital, Duane Road, London W12 OHS.
D
313
314
D. Reichl et al.
Labelled cholesterol introduced into the circulation exchanges with cholesterol in most tissues
of the human body. There is also evidence to suggest that net transport of cholesterol between
plasma and tissues occurs under certain conditions, such as feeding polyunsaturated fats
(Grundy & Ahrens, 1970) or treatment with clofibrate (Grundy, Ahrens, Salen, Schreibman &
Nestel, 1972). Furthermore, since cholesterol is synthesized in the cells of nearly all tissues,
but is removed from the body predominantly by the liver, there must be a continuous flow of
cholesterol from tissue cells to liver, presumably via the blood circulation. These facts point
to the existence of a substantial two-way traffic of cholesterol between plasma and tissues, not
necessarily by the same route in both directions. This raises the question as to how cholesterol
reaches the tissues from the plasma and how it is transported from the tissuesinto the circulation.
Does plasma cholesterol cross the capillary wall as part of intact lipoprotein molecules ? In
what form, whether free or esterified, is cholesterol removed from tissue cells and how is it
transported through the extracellular fluid into the circulation?
Relevant answers to these and other questions could be obtained by detailed analysis of the
lipid and lipoprotein composition of the fluid immediately surrounding tissue cells and by
studying the transport of labelled cholesterol and lipoproteins across the interstitial fluid space.
Unfortunately, it is not practicable to obtain samples of this fluid in quantities sufficient for
lipid and lipoprotein analysis. However, as discussed by Yoffey & Courtice (1970), the
composition of lymph is probably very similar to that of the tissue fluid from which it is
derived. We have therefore begun a study of the transport of cholesterol and of other components of plasma lipoproteins through prenodal lymph obtained from the dorsum of the
human foot. Lymph from this source is uncontaminated by products derived from the intestine
and therefore provides information about lipids that enter the tissue fluids from the plasma
and from the peripheral tissues. A brief account of this work has been given elsewhere (Reichl,
Simons, Mills & Pflug, 1972; Simons, Reichl & Mug, 1972).
METHODS
Clinical details
Observations were made on eight patients with primary hyperlipoproteinaemia and on
three normal subjects. All the patients were under treatment by diet or drugs during the
investigation. Brief clinical details are given in Table 1.
Experimental procedure
In order to obtain information about both early and late phases of the entry of labelled
plasma cholesterol into lymph and tissues, four of the patients were studied within hours of
labelling the plasma and again after intervals of weeks or months. In two other subjects, lymph
was examined only during the early phase after labelling, and in one other only during the
late phase. In four subjects, plasma and tissue biopsies, but not lymph, were obtained during
the late phase after labelling the plasma (see Table 4). Single specimens of lymph for chemical
analysis and for estimation of enzyme activity were obtained from three normal subjects
whose plasma had not been labelled. In most cases, lymph cannulation was begun at about
19.00 hours, 6-7 h after the last meal.
For investigation of the early phase, a prenodal lymph trunk was cannulated. When a steady
flow of lymph was established, the subject was given a single intravenous injection of his or her
Lipids of human peripheral lymph
315
TABLE
1. Clinical details of subjects investigated. Subjects 1-8 ate diets low in animal fat and supplemented
with corn oil throughout the study.
Subject
1
2
3
4
5
6
7
8
9
10
11
Age
(Yea&
Sex
Diagnosis(')
66
35
31
42
F
F
M
19
36
26
30
77
31
F
M
F
F
M
M
IIa
IIa
IIa
IIa
IIb
IIa
IIb
IIa
Normal
Normal
Normal
M
4 0 M
Drugs given during study
D-Thyroxhe+propranolol during first weeks of study
D-Thyroxine+propranolol from day 76 to day 130
Cholestyramine+ clofibrate throughout study
Clofibrate throughout study
Clofibrate throughout study
Cholestyramine until 1 month before study. No drugs thereafter
D-Thyroxine+propranololfrom day 66 to day 233
No drugs during study
No treatment
No treatment
No treatment
~~
~~~
Plasma lipoprotein pattern based on the classification of hyperlipoproteinaemias described by
Beaumont, Carlson, Cooper, Fejfar, Fredrickson & Strasser (1970).
(l)
own plasma labelled with 30-60 pCi of [4-'4C]cholesterol by the method of Lewis & Myant
(1967). Blood samples were taken at suitable intervals after the injection and lymph was
collected continuously for several hours, with sampling intervals of 10-30 min. In one subject,
the intravenous [14C]cholesterolwas given 8 h before cannulation, and in one other the
collection of lymph was discontinued overnight and was resumed on the following day. For
investigation of the late phase, a prenodal lymph trunk was cannulated 29-288 days after
labelling the plasma. Blood and lymph were then sampled as described above. Each patient
gave consent to the intravenous injection of [14C]cholesteroland the minor surgery involved in
lymph-duct cannulation and biopsy of tissues.
Lymph and tissue sampling
A lymphatic trunk on the dorsum of the foot was identified and cannulated essentially by
the method of Kinmonth (1954). About 0.4 ml of a 10% solution of Patent Blue Violet in
water was injected subcutaneously into the dorsum of the first intermetatarsal space. Under
local anaesthesia a longitudinal incision, 3-6 mm long, was made over the blue line which
appeared on the dorsum of the foot within a few minutes of the injection. The lymph trunk
was dissected and cannulated in a retrograde manner with a St Thomas's Hospital pattern
lymphangiography cannula consisting of a 27 standard wire gauge needle attached to a
polythene tube 4-6 cm long and of internal diameter 0.3 mm. The needle was fixed to the
lymph trunk with cat-gut ligatures and the distal end of the cannula was inserted into a glass
vial attached to the subject's foot with adhesive tape. During the collection of lymph the
subject was encouraged to walk.
Biopsies of skin (about 200 mg), adipose tissue (about 0.5 g) and muscle (about 200 mg)
were taken from the calf under local anaesthesia. The specimens were rinsed in physiological
saline (0.15 M-NaCI), dissected free of connective tissue and stored at - 15°C until analysed.
The concentrations of cholesterol in muscle, adipose tissue and skin were approximately
0-5, 1.4 and 1.1 mg/g of fresh tissue respectively. In one subject, the volume of plasma in the
316
D. Reichl et al.
biopsy samples was estimated by measuring the amount of 99Temin the specimen when the
subject had been given an intravenous injection containing 2-5 pCi of 99Te"-labelled human
serum albumin 15 min before the biopsy. In each biopsy, the total volume of plasma was less
than 2 pl/g of fresh tissue.
Extraction and analysis of lipids
Lipids were extracted from plasma, lymph, muscle and adipose tissue by the method of
Folch, Lees & Sloane-Stanley (1957). Before extraction of muscle and adipose tissue, the samples
were homogenized in a glass homogenizer with 1-2 ml of 0.15 M-NaCl. In all cases the chloroform layer containing the extracted lipids was evaporated to dryness and the residue dissolved
in a known volume of chloroform for chemical analysis and radioassay. Skin samples were
cut into small pieces with scissors and extracted once with 10 ml of boiling methanol and
twice with 10 ml of boiling chloroform. The pieces of skin were then ground in a glass homogenizer three times with 20 ml of chloroform-methanol(2 : 1, v/v). The methanol, chloroform
and chloroform-methanol extracts were combined and shaken with 1/5 vol of 0-15 M-NaCI.
The chloroform layer was separated, evaporated to dryness and the residue dissolved in a
known volume of chloroform. A portion of the extract of lymph lipids was taken for separation
into cholesteryl esters, triglycerides, free cholesterol and phospholipids by thin-layer chromatography, as described by Skipski, Smolowe, Sullivan & Barclay (1965). The lipid bands were
located by staining with iodine vapour, scraped from the plate and eluted with chloroform
for subsequent assay. Cholesterol in plasma and lymph was assayed by the method of Clark,
Rubin & Arthur (1968). Plasma triglycerides were assayed by the method of Cramp & Robertson (1968). For assay of lymph triglycerides, the eluates from the thin-layer plates were hydrolysed and the glycerol extracted, as described by Knight & Myant (1970). The glycerol was
assayed by the enzymic method of Garland & Randle (1962). A blank value for each lymph
triglyceride assay was obtained by eluting a band from the thin-layer plate adjacent to the
triglyceride band and taking the eluate through the above procedure. The blank value was
subtracted from the value obtained for the triglyceride band. Lymph phospholipids were
assayed by the method of Chen, Toribara & Warner (1956). Proteins were assayed by the
method of Lowry, Rosebrough, Farr & Randall (1951).
In three subjects, portions of muscle, adipose tissue and skin were hydrolysed in ethanolic
KOH on a steam bath for 3 h and the unsaponifiable lipids extracted from the hydrolysate
with light petroleum. The extracts were submitted to gas-liquid chromatography with a Varian
model 2740 Aerograph, with 1.5% SE-30 supported on Varaport (80-100 mesh) in a 1.82 m
(6 ft) column and with N2 (30 ml/min) as carrier gas. More than 95% of the sterol from each
tissue was eluted as a single peak with the R, of cholesterol.
For measurement of post-heparin lipase activity (PHLA) and cholesterol-esterifying activity
in plasma and lymph, the samples were frozen at - 15°C immediately after collection and
were stored until assayed. PHLA was measured with an emulsion of triglyceride in 5% bovine
serum albumin (pH 8.6) as substrate and with human plasma as activator (Reichl, 1972).
Free fatty acids formed by hydrolysis of the substrate were measured by the method of Novik
(1965). For each assay, tubes containing 0.1 5 M-NaCl instead of lymph or plasma were run in
parallel with the tubes containing the sample to be tested. Cholesterol-esterifying activity in
plasma and lymph was measured either by the method of Glomset & Wright (1964) or by the
method of Stokke & Norum (1971). When the method of Stokke & Norum (1971) was used,
Lipids of human peripheral lymph
317
the lymph or plasma was preincubated with 5,5'-dithiobis-(2-nitrobenzoic acid) for 4 h at 37°C
with shaking and the incubation was continued for a further 1 h after addition of mercaptoethanol.
Ultracentrijiigation
Lymph lipoproteins were separated by preparative ultracentrifugation of 1 ml samples of
lymph in solutions of appropriate density (Havel, Eder & Bragdon, 1955) using an MSE model
60 ultracentrifuge and a 3 x 5 ml swing-out MSE head with 1 ml adaptors. Centrifugation at
105 OOO g was continued for 16 h with solutions of density 1.006 and 1.063 and for 40 h with
solutions of density 1-12.
Immunological methods
Lymph lipoproteins were analysed by the two-dimensional double-diffusion technique of
Ouchterlony (1964) with antibodies to human high-, low-, and very-low-density lipoproteins
(HDL, LDL, VLDL) and lipoprotein-X. Radial immunodiffusion of whole lymph was carried
out by the method of Mancini, Carbonara & Heremans (1965) on agarose plates containing
antibody to human LDL. Serial dilutions of plasma were analysed on the same plate.
Radioassay
For measurement of radioactivity in plasma cholesterol, a portion of the lipid extract was
transferred to a counting vial and evaporated to dryness. The residue was dissolved in 2 ml of
ethanol and 10 ml of toluene containing 0.3% 2,5-diphenyloxazole (POP) and 0.01% 1,4bis-(5-phenyloxazol-2-yl)benzene (POPOP). Radioactivity was determined in a Beckman
LS-250 liquid-scintillation spectrometer with automatic quench correction. Counting rates
were expressed as d.p.m. by comparison with the counting rate of a standard solution of
[14C]toluene assayed under the same conditions. For measurement of radioactivity in lymph
and tissues, a known portion of the lipid extract was transferred to a glass disc and evaporated
to dryness to give an 'infinitely thin' layer. Radioactivity was determined in a Nuclear-Chicago
gas-flow counter with a background counting rate of 1.5 c.p.m. and a counting efficiency of
22%. Counting rates were expressed as d.p.m. by comparison with the counting rate of a
radioactive standard assayed under identical conditions. After radioassay, the lipids on the
planchette were dissolved in chloroform for chemical analysis. In all cases, samples were
counted for long enough to give a statistical counting error of less than +3%.
Materials
[4-14C]Cholesterolwas obtained from The Radiochemical Centre, Amersham, Bucks., U.K.,
and was purified by thin-layer chromatography with benzene-ethyl acetate (10 : 1, v/v) as
solvent. For the preparation of 99Tem,a column containing "Mo (The Radiochemical Centre)
was eluted with 50 ml of 0.15 M-NaCl. Human serum albumin was obtained from The Lister
Institute of Preventive Medicine, London, and was labelled with "Tern by the method of
Jacoby, Arnot, Jeyasingh, Glass & Browne (1969). The lymphangiography set, sterilized by
gamma radiation, was obtained from Macarthys Ltd, Surgical Division, London. Solvents
used for thin-layer chromatography were of AnalaR grade whenever possible. Isoamyl ether,
used for chromatography of lymph lipids, was purified by passage through activated aluminium
oxide. Glycerokinase was obtained from C. F. Boehringer und Soehne, G.m.b.H., Mannheim,
318
D.Reichl et al.
Germany. Antibodies to human HDL and LDL, prepared in rabbits and purified by absorption with human plasma proteins, were obtained from Behringwerke A.G., Marburg-Lahn,
Germany. Antibodies to human lipoprotein-X and VLDL were prepared in rabbits. Lipoprotein-X was prepared from patients with biliary obstruction by the method of Seidel,
Alaupovic & Furman (1969). The VLDL used for raising antibodies was prepared from serum
obtained during the fasting state from a patient with primary hyperprebetalipoproteinaemia.
The serum fraction of density less than 1.006 was prepared essentially by the method of Have1
et al. (1955) and was washed three times by ultracentrifugation in medium of density 1.006.
The final product was dialysed against 0.15 M-NaCl at 4°C. From 10 to 20 mg of lipoprotein-X
or VLDL, in about 1 ml of 0-15 M-NaCl, was mixed with 2 vol. of Freund's adjuvant and injected subcutaneously into rabbits. Several animals were immunized against each antigen.
Trial samples of blood were taken at intervals of 2-3 weeks until a suitable antibody titre
was reached. Larger samples of blood were then drawn for the preparation of serum.
RESULTS
Flow and composition of lymph
A flow of lymph was indicated by the appearance of a column of blue fluid in the cannula
as soon as the needle entered the lymph duct. When the subject's plasma was labelled with
['4C]cholesterol after cannulation, the presence of a direct leak of blood plasma into the
lymph could be excluded by the fact that radioactivity was not detectable in the lymph for at
least 15 min after the injection.
During each cannulation, lymph flow was maintained at a fairly steady rate for up to 3 h,
but the mean rate varied from one subject to another and in the same subject on different
occasions. In nine cannulations performed on six subjects, mean lymph flow varied from
0.25 to 1.96 ml/h. Flow appeared to be related to the amount of exercise taken during the
collection, but was unaffected by intravenous injection of heparin. In all cannulations, lymph
Time after cannulation (min)
FIG. 1 . Lymph flow and protein and cholesterol concentrations in peripheral lymph obtained
during a 2 h cannulation in subject 2. The vertical arrow shows intravenous injection of heparin
(10 i.u./kg). 0, Lymph protein; A , lymph flow; 0 , lymph cholesterol.
Lipids of human peripheral lymph
319
concentrations of total protein and total cholesterol showed little fluctuation and were not
related to lymph flow.
Fig. 1 shows the results of a representative cannulation and Table 2 shows the concentrations
TABLE
2. Lipid composition of plasma and lymph from hyperlipidaemic and normal subjects
Plasma
Subject Day
1
2
3
4
5
6
7
8
9
10
1
156
289
1
170
1
29
225
1
175
1
64
1
288
157
81
1
1
Lymph
Cholesterol Triglycerides Protein
(total)
200
240
326
360
240
320
310
280
280
176
270
250
780
143
120
54
129
183
94
86
72
550
330
50
550
-
215
263
190
214
213
94
123
142
Cholesterol
Total
-
29+3
20
-
2.8f0.1
1.8
2.950.1
3.3 f 0.1
-
30
24
34f 1
42f4
34
25f3
-
2.3 f 0.1
2.7
3.6f0.3
4.1
2.1 f 0.5
2.9
2.6
Triglycerides Phospholipid
Esterltotal
30+2
19
70
42
24
-
Values for cholesterol, triglyceride and phospholipid are expressed in mg/100 ml of plasma or lymph. Values
for lymph protein are expressed in g/100 ml of lymph. Where three or more samples were analysed separately
from a single lymph cannulation, mean valuesf SEM are given.
of total protein and of lipids in the plasma and lymph of all the subjects who were cannulated.
Mean lymph protein concentration varied from 2.1 to 3.6 g/l00 ml. Mean lymph total cholesterol concentration varied from 25 to 70 mg/100 ml and was usually between one-tenth and
one-eighth of the plasma concentration. There was a significant correlation between the
total cholesterol concentration in lymph and that in plasma over a range of plasma cholesterol
concentration from 200 to 780 mg/100 ml (Fig. 2). The ratio of esterified to total cholesterol
in lymph was not significantly different from that in plasma. Lymph triglyceride concentration, measured in the combined lipid extracts from each of five cannulations, varied from
7 to 21 mg/100 ml. There was no correlation between plasma and lymph triglyceride concentration, either in different subjects or in the same subject at different times. In one
hypertriglyceridaemicsubject in whom lymph collection was begun 2 h after a meal, the lymph
triglyceride concentration remained unchanged while the plasma triglyceride concentration
D. Reichl et al.
320
0
100
200
300
400
500
600
700
Plasma cholesterol concentration (mg/ 100ml)
FIG.2. Relation between plasma and peripheral-lymph total cholesterol concentration in thirteen
pairs of samples from seven subjects. Kendall's rank correlation coefficient was +0.62 (P<0.01).
Time (min)
Time (h)
FIG.3. Triglyceride concentration in peripheral lymph (0)
and plasma (0)in a patient (subject 5 )
with type IIb hyperlipoproteinaemia. A lymph cannula was inserted at zero time, 2 h after a meal,
and was withdrawn 3 h later. The cannula was re-inserted for 70 min on the following day. The
patient fasted throughout the test period.
Lipids of human peripheral lymph
FIG.4. Ouchterlony plates demonstrating the presence of plasma lipoprotein antigens in lymph
obtained from the dorsum of the foot. In each of the four quadrants (a, b, c and d), S contained
a fraction of lymph obtained in the ultracentrifuge, 1 contained anti-HDL, 2 contained antiLDL, 3 contained anti-VLDL and 4 contained anti-lipoprotein-X. The density of the lymph
fraction in (a) was less than 1906, in (b) was 1.006-1.063, in (c) was 1.063-1.12 and in (d) was
greater than 1.12.
(Facing p. 320)
Lipids of human peripheral lymph
321
fell from 940 to 470 mg/100 ml (Fig. 3). Lymph phospholipid concentration varied from 8.5 to
22.0 mg/100 ml in three subjects.
Post-heparin lipase activity
Lipase activity was measured in plasma and lymph from subjects 1, 2, 3 and 4. No lipase
activity was detectable in plasma or lymph before intravenous injection of heparin. At 10
min after intravenous injection of heparin, PHLA in the four subjects varied from 80 to
166 pequiv. of fatty acid released/min per litre of plasma. In each patient, PHLA was measured
separately in the pooled lymph collected from 0 to 15 min and from 15 to 30 min after heparin.
PHLA was not significantly different from the blank value in any of the samples of lymph.
To test the possibility that the absence of detectable PHLA in lymph was due to the presence
of an inhibitor, PHLA was measured in samples of plasma taken 10 min after heparin injection,
with and without the addition of an equal volume of post-heparin lymph from the same
subject. No inhibition was observed.
Cholesterol-esterifying activity
Cholesterol-esterifying activity was measured in the lymph and plasma of subjects 2, 3, 10
and 11. The method of Glomset &Wright (1964) was used for subject 2 and that of Stokke &
Norum (1971) was used for subject 3. Lymph and plasma from subjects 10 and 11 were tested
by both methods. Table 3 shows the cholesterol-esterifying activity in lymph from the four
subjects, expressed as a percentage of the activity in an equal volume of serum from the same
subject. In all specimens of lymph tested, esterifying activity was significantly greater than the
TABLE3. Cholesterol-esterifying activity in
lymph and plasma assessed by two methods
Lymph/plasma activity (%)
Subject
Method 1
Method 2
2
3
10
11
8.7
7.9
1.7
2.5
-
6.6
4.3
For method 1 (Glomset & Wright, 1964),
0.1 ml of lymph or serum was incubated for
3 h with 0.9 ml of substrate prepared by
labelling freshly prepared heated serum with
[ 14C]cholesterol. Blank values were obtained
by incubating 0 1 ml of 0 1 5 M-NaCI with
0.9 ml of substrate. For method 2 (Stokke &
Norum, 1971), 0.1 ml of lymph or serum was
pre-incubated for 4 h with [14C]cholesterol in
the presence of enzyme inhibitor and for a
further 1 h after addition of mercaptoethanol.
Blank values were obtained by incubating the
samples for 5 h in the presence of inhibitor but
without addition of rnercaptoethanol.
322
D. Reichl et al.
blank value, but in each subject the activity in lymph was less than 10% of that in plasma.
This finding is in agreement with a single observation reported by Glomset (1968).
Lipoprotein antigens
The quantities of lymph we were able to obtain were not sufficient for analysis of the individual peptides present in plasma lipoproteins. However, several subjects provided sufficient
lymph for qualitative or semi-quantitative tests for the presence of the major antigenic
components of VLDL, LDL and HDL, as defined by Alaupovic (1968). Samples of lymph
from five subjects were tested by the double-diffusion technique of Ouchterlony (1964).
All the samples gave precipitin lines with antibodies to plasma HDL, LDL, VLDL and lipoprotein-X. When lymph and plasma from the same subject were tested on Mancini plates
(Mancini et al., 1965) impregnated with antibody to human LDL, lymph produced a precipitin
halo of the same diameter as that produced by plasma diluted five to eight times, suggesting
that the concentration of apoprotein-B in lymph is one-eighth to one-fifth that in plasma.
Four ultracentrifugal fractions, of densities less than 1.006, 1.006-1.063, 1.063-1.12 and
greater than 1.12, were prepared from the lymph obtained from five subjects and each fraction
was tested on Ouchterlony plates against antibodies to plasma HDL, LDL, VLDL and lipo-
I
I
" 20
I
I
I
40
60
80
A
Time (min)
Day I
Day 156
FIG.5. Specific radioactivity of total cholesterol in plasma (0)and lymph (0)
in subject 1 during
cannulation of a lymph duct in the dorsum of the foot. After a flow of lymph was established
['4C]cholesterol was injected intravenously at zero time and lymph was collected for 70 min.
Single samples of lymph and plasma were obtained 156 days after the injection.
Lipids of human peripheral lymph
323
protein-X. The distribution of lipoprotein antigens in the four fractions was similar in each
sample of lymph. Fig. 4 shows the results obtained with lymph from subject 2. In each quadrant
the lymph was placed in the centre well. The fraction of density less than 1.006 gave no precipitin
lines with any of the four antibodies. The fraction of density 1.006-1-063 gave a single line with
anti-HDL, indicating the presence of apoprotein-A, and a continuous precipitin arc with
anti-LDL and anti-VLDL, indicating the presence of apoprotein-B. The fraction of density
1.063-1.12 gave a continuous arc with anti-LDL and anti-VLDL, indicating the presence of
apoprotein-B. This fraction also gave a precipitin line with anti-lipoprotein-X, which merged
with a second line given with anti-VLDL, showing the presence of apoprotein-C. The fraction
of density greater than 1.12 gave a faint line with anti-HDL. This fraction contained less than
3% of the total cholesterol in whole lymph.
Transport of cholesterol through lymph
Early phase after labelling (day 1). In order to investigate the transport of cholesterolthrough
lymph, the plasma cholesterol was labelled by single intravenous injections of [14C]cholesterol
in three subjects in whom lymph was already draining from a cannula. The specific radioactivity of total cholesterol was then measured in serial samples of plasma and lymph for 80120 min. The results are shown in Figs. 5, 6 and 7. No radioactivity was detectable in the first
15 min sample of lymph collected after the injection. The specific radioactivity of lymph
cholesterol then increased rapidly during the first hour, but showed little further change during
100-
.-"
L
a
a
l
m
20
40
60
Time (mid
Day I
80
100
120
"
Day 170
FIG.6. Specific radioactivity of total cholesterol in plasma (0)and lymph (0) in subject 2 during
lymph-ductcannulation.After a flow of lymph was established,[ 14C]cholesterolwas injected intravenously at zero time and lymph was collected for 2 h. Single samples of lymph and plasma were
obtained 170 days after the injection.
-1
40
80 120
Time ( m i d
40
120 160
Time ( m i d
80
Day 225
Day 29
Day I
FIG.7. Specific radioactivity of total cholesterol in plasma (0)and lymph ( 0 ) in subject 3 during
lymph-duct cannulation. After a flow of lymph was established, [14C]cholesterolwas injected at
zero time and lymph was collected for 2 h. The cannula was re-inserted 29 days after the
injection and samples of plasma and lymph were obtained for 2) h. Single samples of plasma
and lymph were also obtained 225 days after the injection.
Time ( h l
Day I
Day 175
FIG.8. Specific radioactivity of total cholesterol in plasma (0)and lymph ( 0 ) in subject 4 during
lymph-duct cannulation. [ 14C]Cholesterolwas injected intravenously at zero time. At 8 h later
(shown by the arrow) a lymph duct was cannulated and two samples each of plasma and lymph
were obtained. Single samples of plasma and lymph were also obtained 175 days after the
injection.
325
Lipids of human peripheral lymph
the second hour. In the last sample of lymph obtained during the early phase in these three
subjects, the specific radioactivity of lymph cholesterol was less than 10% of that of plasma
cholesterol at the same time. In subject 4 the procedure was modified by labelling the plasma
~ith[~~C]cholesterol8
h before insertion of the cannula, so that alater stage in the equilibration
between plasma and lymph cholesterol could be examined. The specific radioactivity of lymph
cholesterol collected during the ninth and tenth hour was less than 30% of that of the plasma
cholesterol (Fig. 8).
Late phase after labelling. The slow rate of equilibration between plasma and lymph cholesterol during the first few hours after lymph cannulation would be expected if a proportion of
TABLE4. Lymph/plasma and tissuelplasma cholesterol specific radioactivity ratios during late phase after intravenous injection of [‘4C]cholesterol
Subject Day
3
5
29
8
81
1
7
156
157
2
170
4
175
3
225
64
1
288
6
288
Plasma
Specific radioactivity ratio(l)
cholesterol
Cholesterol
specific
Adipose
fraction
radioactivity Lymph Muscle tissue Skin
(d.p.m./mg)
-
-
-
-
2.27
2.84
0.62
1.21
1.27
-
-
3.83
2.38
6.21
0.85
1.46
5.82
8.64
614
2.36
2.37
-
43
7.86
-
0.95
1.06
0.74
0.85
1.05
0.12
1.05
0.48
050
0.39
1.16
1.31
0.73
1.90
1.91
15
5.2
11.7
3.9
-
1.09
1.19
-
0.61
0.80
0.13
1.33
1.47
0.30
2.57
3.94
4.46
1.61
1.61
1.80
0.26
2.83
3.23
3.10
3.26
6.56
6.17
2.58
-
-
Total
Total
Free
Ester
Total
Free
Ester
Total
Total
Free
Ester
Total
Free
Ester
Total
Free
304
308
Total
Free
Ester
Free
Ester
Total
Free
Ester
331
151
83
137
27
24
1.06
-
10.95
23.42
5.94
-
2.70
-
-
4.8
4.5
1.44
1.20
-
-
( l ) Each value is the ratio of the specific radioactivity of cholesterol (total,
free or esterified) in lymph or tissues to that of total cholesterol in plasma on
the same day.
326
D. Reichl et al.
the cholesterol in lymph is not derived directly from plasma but is contributed by the tissues.
To test this possibility, cholesterol in lymph, plasma and tissues was examined at relatively
long intervals after ['4C]cholesterol administration, when the specific radioactivity of tissue
cholesterol may be several times that of plasma cholesterol (Moutafis & Myant, 1969). In
five subjects ( 1 , 2 , 3 , 4 and 6) single samples of lymph, plasma and tissue were obtained between
days 156 and 288 after the injection of [14C]cholesterol. In subjects 1, 5, 7 and 8 samples of
plasma and tissue, but not of lymph, were obtained between days 64 and 288. In subject 3,
samples of lymph. and plasma were obtained on day 29, this subject providing lymph and
plasma for analysis on three occasions (days 1, 29 and 288). Whenever sufficient lymph or
tissue was available, specific radioactivity was measured in total, free and esterified cholesterol.
Since plasma free and esterified cholesterol have almost identical specific radioactivities
5-7 days after administration of ['4C]cholesterol (Hellman, Rosenfeld, Eidinoff, Fukushima,
Gallagher, Wang & Adlersberg, 1955), plasma specific radioactivity during the late phase was
measured only in total cholesterol.
Figs. 5 , 6, 7 and 8 show the specific radioactivities of total cholesterol in lymph and plasma
during the late phase in the first four subjects. Table 4 shows the ratio of the specific radioactivity of cholesterol in lymph or tissues to that in plasma in eight subjects on ten separate
occasions between day 29 and day 288. On day 29, specific radioactivity of total cholesterol in
lymph was the same as that of plasma total cholesterol, but in the five samples of lymph
obtained from day 156 to day 288 the specific radioactivity of total cholesterol was 2-1 1 times
that in plasma. In the three samples of lymph large enough for separate analysis of free and
esterified cholesterol, the specific radioactivity of free cholesterol was 3-9-7.3 times that of
esterified cholesterol in lymph and was 6-2-23.4 times that of total cholesterol in plasma.
In six of the seven muscle biopsies taken between day 64and day288, the specificradioactivity
of total cholesterol was 1.2-5.8 times that of plasma cholesterol. In both the muscle biopsies
in which free and esterified cholesterol were analysed separately, free cholesterol had a higher
specific radioactivity than esterified cholesterol. However, on no occasion did the specific
radioactivity of muscle cholesterol, either free or esterified, exceed that of the corresponding
cholesterol fraction in lymph.
In the eight samples of adipose tissue taken after day 64, total cholesterol had a specific
radioactivity 1.3-3.9 times that of plasma cholesterol. In five of the six adipose tissue samples
in which a comparison was possible, the specific radioactivity of free cholesterol was 1-1-6.9
times that of esterified cholesterol. However, on every occasion on which adipose tissue and
lymph cholesterol were analysed at the same time, the specificradioactivity of lymph cholesterol
was higher than that of the corresponding cholesterol fraction in adipose tissue.
The results obtained from normal skin were rather inconsistent. On two out of six occasions,
skin total cholesterol had a higher specific radioactivity than plasma cholesterol. As in lymph,
muscle and adipose tissue, free cholesterol of skin had a higher specific radioactivity than
esterified cholesterol, but on only one occasion (subject 3, day 225) was the specific radioactivity of skin cholesterol above that of the corresponding cholesterol fraction in lymph.
DISCUSSION
In agreement with earlier work on peripheral lymph in animals (see Yoffey & Courtice, 1970),
lymph draining from the human foot contains cholesterol, phospholipids and triglycerides,
Lipids of human peripheral lymph
327
although the concentration of each lipid in lymph is much lower than that in plasma. The
positive correlation between lymph and plasma cholesterol concentration would be expected
if a significant fraction of lymph cholesterol is derived directly from the plasma. The rapid
appearance of radioactive cholesterol in lymph after intravenous injection of [14C]cholesterol
also indicates that cholesterol molecules can pass from the circulation directly into lymph or
into the fluid space drained by lymphatic ducts. However, this may reflect exchange of cholesterol across the walls of the blood capillaries, rather than net flow from plasma to lymph. The
question whether cholesterol can cross the capillary wall in both directions or whether, having
once entered the extravascularfluid space, it can only return to the circulation via the lymphatic
channels, cannot be answered by the results of the present work. The triglyceride present in
lymph may also be derived directly from the plasma. The presence of lipoprotein lipase in
capillary endothelium may allow only a small quantity of plasma triglyceride to escape
hydrolysis during its passage through the walls of the capillaries, and hence could explain
the lack of correlation between lymph and plasma triglyceride concentrations. On the other
hand, it is possible that some or all of the triglyceride in lymph is derived from extravascular
tissues.
Courtice & Morris (1955) detected a- and B-lipoproteins in peripheral lymph from cats
by paper electrophoresis and concluded that intact lipoproteins could be transferred from
plasma to lymph. However, the observation that lipoproteins in lymph and plasma have the
same electrophoretic mobility does not prove that their lipid composition is identical. Our
immunochemical findings show that lymph from the human foot contains the three major
antigens of plasma lipoproteins. This is compatible with the passage of plasma lipoproteins
across the capillary walls. However, the distribution of the A, B and C apoproteins in the
three ultracentrifugal fractions of lymph suggests that, even if some lipoprotein does reach
the lymph intact, the greater part undergoes considerable modification after it has left the
circulation. In particular, our finding that all the apoprotein-C and part of the apoprotein-B
in lymph is present in the fraction of density greater than 1.063 suggests that these apoproteins
lose much of their lipid load before reaching peripheral lymph.
Our failure to detect lipoprotein lipase in lymph, either before or after intravenous injection
of heparin, is consistent with the current view that hydrolysis of plasma triglyceride takes place
at the luminal surface of the capillary endothelium or within the walls of capillaries (Robinson,
1970; Blanchette-Mackie & Scow, 1971). However, if lipoprotein lipase in capillary endothelium is synthesized in the tissues (Robinson, 1970), it is perhaps surprising that the enzyme
does not enter lymphatic channels in detectable amounts during its passage from tissue cells
to the luminal surface of the blood capillaries.
As discussed above, the early appearance of radioactive cholesterol in peripheral lymph
after intravenous injection of [14C]cholesterolindicates direct transfer of cholesterol from
plasma to lymph, whether by exchange or mass transport. However, at intervals of 156 days
or more after labelling of the plasma cholesterol the specific radioactivity of total cholesterol
in lymph was several times that in plasma and the specificradioactivity of lymph free cholesterol
was higher than that of lymph esterified cholesterol.This shows that some of the cholesterol in
lymph is not derived directly from plasma but from a pool of tissue cholesterol which acquires
a higher specific radioactivity than that of the plasma cholesterol at long intervals after a single
intravenous injection of radioactive cholesterol. Moutafis & Myant (1969) have shown that,
owing to the presence of pools of cholesterol with slow turnover, the speczc radioactivity of
328
D.Reichl et al.
total cholesterol in skin exceeds that in plasma several weeks after labelling the plasma
cholesterol. This observation has been confirmed by Samuel, Perl, Holtzman, Rochman &
Lieberman (1972), and is shown in the present work to be true for muscle and adipose tissue,
as well as for skin. In only one case was the specific radioactivity of free or esterified cholesterol
in any of the three tissues examined during the late phase as high as that of the corresponding
cholesterol fraction in lymph. However, this does not exclude any of these tissues as a source
of the lymph cholesterol of high specific radioactivity, since there may be several pools of
cholesterol with different rates of turnover in each tissue. If so, there could be pools of cholesterol in each tissue with higher specific radioactivity than that of the cholesterol extracted from
the whole tissue.
The free cholesterol of high specific radioactivity obtained from lymph during the late phase
may be derived from cholesterol that has become incorporated into cell membranes, since
membrane cholesterol is predominantly unesterified. If so, the cells drained by the lymphatics
in the dorsum of the foot must contain membrane cholesterol, derived ultimately from the
plasma, whose rate of turnover is so slow that its specific radioactivity becomes several times
higher than that of the plasma cholesterol at very long intervals after intravenous injection of
['4C]cholesterol. Presumably, cholesterol that leaves the tissues to enter lymph is incorporated
into lipoprotein, though we have no evidence as to which apolipoproteins of lymph are
primarily responsible for accepting cholesterol from tissue cells.
The presence, during the late phase, of cholesteryl esters in lymph with specific radioactivity
higher than that of the plasma cholesteryl esters raises the possibility that some of the lymph
esters arise by esterification of free cholesterol within lymph itself or in the extracellular fluid
draining into lymph channels. This would be in keeping with the scheme suggested by Glomset
(1 968), according to which free cholesterol in extravascular tissues is taken up by lipoproteins
in the extracellular fluid and is then esterified by lecithin-cholesterol acyl transferase. As
shown in the present work human peripheral lymph exhibits cholesterol-esterifying activity.
However, our observations provide no evidence that this is due to the presence of an enzyme
identical with the acyl transferase of plasma.
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