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Leucine Turnover in Patients with Nephrotic Syndrome:
Evidence Suggesting Body Protein Conservation
VICTORIA S. LIM,* MARSHA WOLFSON,� KEVIN E. YARASHESKI,�
MICHAEL J. FLANIGAN,* and JOEL D. KOPPLE�’*Departments of Medicine, University of Iowa, Iowa City, Iowa; ‘�Oregon Health Science University and
Portland VAMC, Portland, Oregon; �Washington University, St. Louis, Missouri, and �Harbor-UCLA Medical
Center, Los Angeles, C’alifcrnia.
Abstract. Whole-body leucine flux was measured in eight
patients with nephrotic syndrome and in five healthy subjects
by primed-constant infusion of L-[l-’3C leucine]. Plasma en-
richment of ‘3C leucine and ‘3C a-keto-isocaproate (13C KIC)
was measured by gas chromatography/mass spectrometry, and
expired � 3C02 was measured by isotope ratio mass spectrom-
etry. Leucine kinetics, calculated from the primary pool en-
richment [‘3C leucine], showed no difference between the
nephrotic patients and the control subjects. Kinetics derived
from the reciprocal pool [ 1 - I 3C KIC] enrichment, however,
showed that leucine turnover rates were reduced in the ne-
phrotic patients. The values (�tmobIkg per h, means ± SD)
comparing the patients and the control subjects are as follows:
rate of leucine release from protein degradation, 99 ± 6 and
I 17 ± 12 (P = 0.007); leucine oxidation rate, 15 ± 7 and 22 ±
3 (P = 0.04); rate of leucine incorporation into body protein
[5], 84 ± 10 and 95 ± 6 (P = 0.04); protein turnover rate,
3.99 ± 0.49 and 4.72 ± 0.25 glkg per d (P = 0.007). Nitrogen
balance, measured only in the nephrotic patients, showed a
mean positive balance of 0.5 g/d. In the nephrotic and control
subjects, protein intake levels were 0.84 ± 0. 16 and 1 .1 7 ±
0. 1 8 glkg per d (P = 0.002), respectively, and energy intake
levels were 33.3 ± 8.5 and 33.9 ± 2.4 kcallkg per d, respec-
tively. Linear correlations between leucine turnover rates and
protein intake were highly significant. This study found that
nephrotic patients given a modestly protein-restricted diet were
able to maintain positive nitrogen balance. Moreover, leucine
flux measurements showed downregulation of protein degra-
dation and amino acid oxidation, reflecting appropriate adap-
tation to a lower protein intake. (J Am Soc Nephrol 9: 1067-
1073, 1998)
Patients with nephrotic syndrome often exhibit protein-energy
malnutrition due to increased protein loss into the urine and
enhanced in situ renal protein degradation (1 ,2). Hypoalbumin-
emia and low total serum protein concentration indicate vis-
ceral protein depletion. Studies of albumin metabolism indicate
a reduced rate of albumin catabolism (3-5), but little is known
about whole-body protein metabolism outside of the albumin
pool. The observations of muscle wasting and subnormal body
anthropometry suggest that somatic protein depletion may oc-
cur simultaneously. On one hand, it has been hypothesized that
body protein outside of the albumin compartment may have
augmented proteolysis to provide substrate for increased he-
patic albumin synthesis. On the other hand, it is also possible
that protein metabolism may be downregulated in an effort to
conserve body protein because of protein deficiency. The
present study was designed to assess protein turnover in pa-
tients with nephrotic syndrome by measuring whole-body
leucine flux.
Received May 5, 1997. Accepted November 26. 1997.
Correspondence to Dr. Victoria S. Lim, Department of Internal Medicine,T310, GH, University of Iowa Hospitals, 200 Hawkins Drive, Iowa City, IA
52242.
1046-6673/0906- l067$03.00/0
Journal of the American Society of Nephrology
Copyright 0 1998 by the American Society of Nephrology
Materials and MethodsExperimental Design
Whole-body leucine flux was measured in eight subjects with
nephrotic syndrome. These patients were enrolled in a double-blind,
placebo-controlled study to assess the effect of fosinopril, an angio-tensin-converting enzyme inhibitor, on proteinuria, nitrogen metabo-
lism, and metabolic parameters in patients with nephrotic syndrome.
Each subject stayed in the Clinical Research Center (CRC) for 35
consecutive days while consuming a constant diet. The first 15 d
served as the control period, the last 20 d, the experimental period,
during which time the subjects were given either a placebo or fosi-
nopril. Before the study, each subject visited the CRC dietitian for a
dietary interview, followed by a 3-d dietary recording. From this
information. a meal plan was designed for each subject. The goal of
the diet was to supply 0.9 g of protein and 35 kcal of energy per
kilogram of desirable body weight each day. Desirable body weight
was determined from the Metropolitan Life Insurance Company Stan-
dards (6). Within these parameters, the diet was modified to fit the
preferences and the usual eating habits of the study subjects. Intake of
sodium chloride was limited to 5 g/d. Two subjects with non-insulin-
dependent diabetes were placed on a diabetic diet as well. Nitrogen
balance studies were performed as described elsewhere (7,8). Body
weight, serum urea nitrogen, and creatinine, as well as 24-h urine
excretion of total nitrogen, urea nitrogen, creatinine, total protein, and
albumin. were measured daily. Feces were collected continuously in
5-d periods. Duplicate diet (prepared weekly), rejected food, emesis,
and pooled feces were each analyzed for total nitrogen. Nitrogen was
measured by spectral chemiluminescence using an Antek model 720
1068 Journal of the American Society of Nephrology
nitrogen detector (Houston, TX). Routine serum chemistries, blood
counts, and serum lipid profiles were measured every fifth day.
Additionally, whole-body leucine flux was measured twice, once on
day 15 and again on day 35, in patients housed at the University of
Iowa Hospitals in Iowa City, Iowa, and the Veterans Administration
Hospital in Portland. Oregon. The nitrogen balances represent ad-
justed total daily balances and were calculated by the following
equations: Nb = N.� - EN1,rir,c + � + z�urea N pool], Z�urea N
pool = (SUN1 - SUN) X BW x 0.60 + (BW1 - BW1) X SUNS,
where SUN is serum urea nitrogen (gIL). BW is body weight (kg), and
i and f represent the initial and the final time points of the collection
periods (9,10). The current report focuses on the studies of leucine
flux. It includes a total of 12 leucine tracer studies; eight were
measured on day 15 of the baseline period and four on day 35 during
placebo treatment.
Five healthy subjects were recruited for leucine flux measurements.
These subjects ingested an unrestricted but constant diet prepared by
the CRC for 6 d before the study, and leucine flux was measured after
a 12-h fast on the seventh day. As with the nephrotic patients, the
healthy subjects had a dietary interview and a 3-d dietary recording
from which a meal plan was prepared for each individual.
Leucine Flux Measurement
L-[l-’3Clleucine and NaH’3C03, respectively, 99 and 95 atom
percent enrichment, were purchased from Mass Trace (Woburn, MA).
Leucine kinetics were measured by a primed-constant infusion tech-
nique during substrate and isotopic steady state after a 12-h fast. The
priming dose consisted of 50 mg of L-[l-’3C]leucine and 0.11 mg/kg
NaH’3CO3, and the sustaining infusion, L-[l-’3C ] leucine, at 50 mg/h
for 4 h. Infusates were prepared aseptically on the afternoon before the
experiment. ‘3C enrichment of plasma leucine and plasma a-keto-
isocaproate (KIC) was measured by gas chromatography mass spec-
trometry, and 13C0, enrichment in the expired breath was measured
by isotope ratio mass spectrometry before and every 30 mm after the
initiation of L-[1 -‘ 3C]leucine infusion, as described previously
(11,12).
Leucine flux (Q) denotes movement of leucine into (Rate of ap-
pearance, R�) and out of (Rate of disappearance, Rd) the leucine pool.
In steady state, K = Rd.
FE 1
Q= [E� 1]
where E. is ‘3C leucine enrichment in the infusate (100%), E� is ‘3C
leucine or 13C KIC enrichment in the plasma at isotopic equilibrium,
and i is L-[l-’3Cileucine infusion rate (p.mol/kg per h). Leucine
appearance rate into the plasma leucine (primary) pool was calculated
from plasma ‘3C leucine enrichment, whereas leucine appearance rate
into the whole-body leucine (reciprocal) pooi was derived from
plasma 13C KIC enrichment (13,14).
The rate of I 3C0, release from tracer leucine oxidation was cal-
culated as follows:
fFCO� x ECO�\ /60 X 41.6F’3C02 � BW �) X � 100 � 0.81
where FCO, is the CO2 production rate, ECO2 refers to ‘3C0,
enrichment in the expired gas at isotopic steady state, and BW is body
weight (kg). The constants 60 (mm/h) and 41.6 (�molIml at standard
temperature and pressure) convert FCO2 from ml/min to �mol/h. The
factor 100 changes atom percent excess from a percent to a fraction,
and the factor 0.8 1 represents the fraction of ‘ 3CO2 produced by
L-[l-’3C] leucine oxidation released from the body bicarbonate pool
into the expired breath (15).
The rate of leucine oxidation (C) is then calculated as:
C�F’3CO2[�--�-] x 100
Quantification of amino acid metabolism was based on the rela-
tionship stating that Q = B + I = S + C, where Q is flux or total
turnover rate, B represents the rate of amino acid release from endog-
enous protein breakdown, and I is the rate of exogenous intake of the
amino acid. S is the rate of amino acid incorporation into protein or the
rate of protein synthesis, and C is the rate of amino acid oxidation. In
the postabsorptive state, I = 0 and B, therefore, equals Q. In general,routes of nonoxidative leucine disposal other than S are assumed to be
negligible (16,17).
CO2 Production Rate and Collection of Expired Gas
CO2 production rate (FCO2 or VCO2) was determined by a porta-
ble metabolic gas monitor (MGMII, Utah Medical Products, Midvale,
UT). Before the study, the procedure for the FCO2 measurement wasexplained to each subject, who was then given sufficient time to
become familiar with the equipment. During measurement, a mouth-
piece attached to a two-way valve was used. Expired gas was sampled
by a small gas line leading from the mouthpiece to an infrared CO2
sensor. Simultaneously, tidal volume and frequency of respiration
were recorded by a pneumotachograph, and expired ventilation was
measured by an ultrasonic flow transducer. The partial pressure of the
inspired CO2 was taken from that of the atmosphere. FCO2 was
calculated by standard equations and corrected for standard tempera-
ture and pressure, dry. Before each experiment, the equipment was
calibrated with a standard reference gas consisting of 10% CO2, and
the flow transducer was checked by a calibrated syringe. During each
measurement, the expired gas was collected in a Douglas bag, and a
sample was transferred anaerobically into sealed vacuum tubes
(Venoject, Terumo Medical, Elkton, MO) for quantification of ‘3CO2
enrichment by isotope ratio mass spectrometry (18). Measurements
and sampling were taken before and every 30 mm after the initiation
of L-[ I - I 3C]leucine infusion. Each measurement and collection lasted
5 mm.
Calculation of Protein Flux
Protein flux, or Qprotcin’ was derived as follows:[ (QKICx 131
24
0.078
where I 3 1 is the molecular weight of leucine, 24 converts the unit
from hours to days, 106 converts micrograms to grams, and 0.078 is
taken from the general estimate that whole-body protein contains an
average of 7.8% leucine (19).
Statistical Analyses
For each leucine flux, nine sets of raw data were available, onebefore and eight after the initiation of L-[1-13C]leucine infusion at
30-mm intervals. The raw data consisted of plasma ‘3C leucine and
‘3C KIC, and breath ‘3C0, enrichment (duplicates measured for each
breath) and CO, production rate. From these values, rates of protein
degradation and synthesis, and leucine oxidation were derived.
Plasma ‘3C leucine and 13C KIC enrichment were generally nonde-
tectable before tracer infusion. Breath ‘3C02, however, was not zero
Leucine Turnover in Nephrotic Patients 1069
because of the natural 13C abundance in the food, and oxidation of
such food resulted in trace amounts of ‘3C0, in the breath before
L-[ I - I 3Clleucine infusion. The zero time breath enrichment was then
subtracted from the enrichment obtained at each time point after tracer
infusion. The 30-mm values were excluded because they have not
quite reached isotope steady state. Plasma and breath isotopic enrich-ment from 60 to 240 mm, assessed by ANOVA, showed no difference
among the different sampling times. Thus, for each leucine flux study,
the mean value from the seven data points was used. All values are
then presented as means ± SD for the nephrotic and the control
groups. Statistical differences between the two groups were assessed
by a’ test. Relationships between leucine kinetics and the variousclinical parameters were tested by linear regression analysis and
correlation coefficients.
Institutional Review Board Approval
The protocol was approved by the Institutional Review Board ofthe participating centers, and informed consent was obtained from
each study subject.
ResultsTable 1 summarizes the demographics and dietary intake of
the nephrotic patients and the healthy control subjects. The
patient group consisted of six men and two women, and their
ages ranged from 3 1 to 78 yr. Four patients had membranous
nephropathy, two had focal segmental glomerulosclerosis, and
two had non-insulin-dependent diabetes mellitus. Their mean
age of 56.6 ± 16.2 yr and mean body mass index of 26.8 ± 4.7
kg/m2 were not different from those of the control subjects
(50.2 ± I 2.0 yr and 27.0 ± I .8 kg/rn2, respectively). Protein
intake among the patients ranged from 0.69 to 1 .13 glkg per d,
and the mean value was 0.84 ± 0. 16 glkg per d. Energy intake
varied from 23.9 to 50.0 kcallkg per d, and the mean value was
33.3 ± 8.5 kcal/kg per d. In this project, we measured 12
leucine fluxes in eight nephrotic patients, i.e. , four patients
underwent two studies. The second set of studies ( I a, 2a, 6a,
and 8a) was done during the experimental period while the
patients were taking a placebo. We have treated each leucine
flux measurement as an independent study, because the mea-
surements were conducted 20 d apart. In the control subjects,
the mean protein and energy intake were, respectively, I .I 7 ±
0.18 glkg per d and 33.9 ± 2.4 kcal/kg per d. Protein intake,
but not energy intake, was significantly higher in the control
subjects (P = 0.002). Daily nitrogen balance (which was
adjusted for change in body urea nitrogen) in the patients
ranged from - 1 .45 to 2.67 g/d, with a mean positive value of
Table 1. Demographics and dietary intake of the nephrotic patients and the healthy control subjects�’
Subject Gender/Age DxHt
(cm)Wt
(kg)BMI
(kg/m2)Prot�
(g/kg per d)Energy,
(kcal/kg per d)NB
(g/d)
Patient
1 M/35 Memb 178 61.1 19.3 1.13 50.0 0.11
la 62.0 19.6 1.11 49.3 -0.29
2 M/6l FSG 182 85.0 26.2 0.81 28.9 0.50
2a 84.4 25.5 0.83 29.7 -0.21
3 M!78 Memb 166 63.6 22.9 1.01 39.4 1.15
4 F/56 FSO 151 56.7 25.1 0.86 33.1 0.70
S M/6l DM11 174 98.4 32.5 0.71 23.9 1.04
6 M/3l Memb 180 100.3 31.0 0.69 27.9 1.00
6a 99.4 30.7 0.69 28.1 2.67
7 M/7 1 Memb 1 70 97.0 33.6 0.69 26.7 - I .45
8 F/60 DM11 173 84.5 28.1 0.80 31.1 0.62
8a 82.5 24.4 0.82 31.8 0.17
mean ± SD 56.6 ± 16.2 26.8 ± 4.7 0.84 ± 0.16 33.3 ± 8.5 0.50 ± 1.00
Control
I M!47 182 80.0 24.2 1.13 31.0
2 M/66 171 84.8 29.0 1.24 33.1
3 M/47 162 69.2 26.5 1.12 37.3
4 F/34 168 77.4 27.6 1.42 35.2
S M!57 178 87.8 27.7 0.93 33.1
mean ± SD 50.2 ± 12.0 27.0 ± 1.8 1.17 ± o18b 33.9 ± 2.4
a The eight patients are listed as numbers I through 8, and I a, 2a, 6a, and 8a were the subjects who had leucine flux measured a second
time. The five control subjects are listed as numbers 1 through 5. BMI = Wt (kg)/Ht (m)2. NB N1� - [Nurine + � + �urea N
pool], and �urea N pool (SUNs. SUN1) X BW1 X 0.6 + (BW, BW1) X SUNf, where SUN is serum urea nitrogen (gIL), BW is bodyweight (kg) and i and f represent the initial and the final time points. Dx, disease; Ht, height; Wt, weight; BMI. body mass index; Prot�,
protein intake; Energy1�, energy intake; NB, nitrogen balance; Memb, membraneous nephropathy; FSG, focal segmental
glomerulosclerosis; DM11, non-insulin dependent diabetes mellitus.b Statistically significant difference between the control subjects and the patients.
6.0-
#{149}�C0EaI-C
Lu‘Up
a00
E1.0
180 240
1070 Journal of the American Society of Nephrology
0.5 ± 1 .0 g/d. If the nitrogen balances were adjusted for
unmeasured nitrogen losses from skin, respiration, and blood
drawing (which probably average approximately 0.5 g/d), the
nitrogen balance would be neutral.
Table 2 lists the proteinuria, creatinine clearance, serum
albumin, and serum lipid profile of the nephrotic patients. The
mean urinary protein excretion was 7.59 ± 5.46 g/d and the
mean creatinine clearance was 64.2 ± 34.0 mi/mm. Serum
albumin was significantly reduced, with a mean value of
3.02 ± 0.46 g/dl. Both serum triglycerides and total cholesterol
were elevated to mean values of 374 and 32 1 mg/dl, respec-
tively.
Figure 1 plots the mole percent enrichment of plasma ‘3C
leucine and ‘3C KIC and breath ‘3CO2 from 60 to 240 mm
during infusion study in the nephrotic patients showing mini-
mal fluctuation of the enrichment, indicating achievement of a
steady-state condition.
Table 3 summarizes leucine kinetics in the nephrotic and the
control subjects. There are two sets of data: one set is derived
from plasma enrichment of ‘3C leucine, which represents the
primary leucine pool kinetics, and the other set, from plasma
enrichment of ‘3C KIC, is the reciprocal pool kinetics. On the
basis of data obtained from the primary poo1 calculations,
leucine kinetics were not different between the nephrotic pa-
tients and the control subjects. Data derived from the reciprocal
pool, however, showed that all parameters of leucine turnover,
including leucine release from endogenous protein breakdown,
leucine oxidation, and leucine incorporation into protein, were
significantly reduced in the nephrotic patients. The ‘3CO2production rate was not different between the healthy control
subjects and the patients. VCO, values, which are not listed in
Table 3 (240 ± 82 and 227 ± 37 mI/mm, respectively, for the
patients and control subjects) also were not different. Protein
flux, calculated from leucine appearance into the total body
leucine pool, was lower in the nephrotic patients than in the
I�I�1L��5.0 �
4.0
. Plasma 13Cku
�A- Plasma 13C KIC
. Bmtk 13C02
05 ‘ � �. 60 120
Time ( mm)
Figure 1. Plasma 3C-leucine U and ‘3C a-keto-isocaproate (‘3C-KIC) A and breath I 3CO, #{149}enrichment during leucine flux measure-
ment, showing achievement of steady-state condition in the nephroticpatients. None of the values for each group listed from 60 to 240 mm
was different, as measured by ANOVA. Actual values for breath
13CO2 enrichment are the plotted values X l02.
control subjects (3.99 glkg per d versus 4.72 glkg per d [P
0.007]).
Linear regression analysis between quantitative urinary pro-
tein excretion and leucine turnover parameters failed to dis-
close any significant correlation. Furthermore, there also was
no significant correlation between urine protein excretion and
Table 2. Proteinuria, renal function, and metabolic profile of the nephrotic patientsa
PatientU rolcin
(‘�/d)Ccr
(ml/min)BUN
(mg/dl)5cr
(mg/dl)5albumin
(g/dl)5TG
(mg/dl)5chol
(mg/dl)
1 3.01 70.8 15 1.4 2.95 180 234
Ia 3.21 70.4 14 1.4 2.91 159 235
2 8.22 53.2 37 2.1 2.78 251 338
2a 7.90 54.7 35 2.0 2.79 310 319
3 5.95 30.0 31 2.2 2.31 220 361
4 3.40 41.0 21 1.5 2.51 453 530
5 5.02 69.9 12 1.1 2.53 115 226
6 4.06 129.0 10 1.1 3.38 186 320
6a 3.39 126.0 10 1.1 3.49 179 243
7 7.25 70.1 15 1.1 3.30 284 327
8 20.54 29.1 24 3.7 3.51 1041 358
8a 15.39 26.5 27 3.7 3.72 1113 361
mean ± SD 7.59 ± 5.46 64.2 ± 34.0 21 ± 10 1.9 ± 0.9 3.02 ± 0.46 374 ± 340 321 ± 85
a Upr�icin� urine protein; Ccr, creatinine clearance; BUN, blood urea nitrogen; �cr’ serum creatinine; 5albumil’ serum albumin; 5TG’ serum
triglyceride; 5chol’ serum cholesterol.
Leucine Turnover in Nephrotic Patients 1071
Table 3. Leucine appearance and disposal and protein flux in nephrotic patients and healthy subjects�’
Group Ft
Ra Breakdown
(Q)(j.tmol/kg per h)
Ql��/QKlcF’3C02
(p.mollkg per h)
Rd
Qprotein
(g/kg per d)Oxidation Synthesis
(C) (5)
(�mo1/kg per h)
P-Leu 12 84.5 ± 9.6 12.4 ± 4.8 72.1 ± 9.9
C-Leu S 89.1 ± 4.1 16.8 ± 2.0 72.3 ± 5.0
P-KIC 99.1 ± 12.P’ 0.86’ 0.704 ± 0.295 14.8 ± 6.8c 84.3 ± 9.6’ 3.99 ± 0.49c
C-KIC 1 17.0 ± 6.3 0.76 0.792 ± 0.101 22.1 ± 3.0 95.0 ± 5.8 4.72 ± 0.25
a P-Leu and C-Leu indicate that data in the corresponding rows were derived from plasma 13C leucine enrichment (primary pool).
respectively, of patients and control subjects. Similarly, P-KIC and C-KIC represent data calculated from ‘3C KIC enrichment,
representing the reciprocal pool kinetics, in the patients and the control subjects. R�, and Rd. leucine appearance and disposal rates,
respectively. R�,, leucine derived from protein breakdown; in the absence of exogenous protein intake, it is also equal to total body leucine
flux, or Q. Rd �S the sum of leucine oxidation and leucine incorporation into protein. F’3CO2 = ‘3CO2 production rate. Q1,r(,)cj) or proteinflux is derived as follows: [(QKLc X 131 X 24/106)/0.0781, where 131 is molecular weight of leucine, 24 converts the unit from hours to
days, lO� converts micrograms to grams, and 0.078 is taken from the general estimate that animal protein contains an average of 7.8%leucine. Leucine kinetics from the reciprocal, as well as the primary. pool in patients were compared with those of the control subjects
using the t test.bp < 0.01.
cP < 0.05.
Table 4. Linear regressions between protein and energy intake on leucine turnover
Kinetic Parameter Protein Intake Energy Intake
Leucine from protein degradation R = 0.72 P = 0.001 R = 0.50 P = 0.042
Leucine oxidation R = 0.50 P = 0.043 R = 0.32 P = 0.2 1 1
Leucine to protein synthesis R = 0.65 P = 0.005 R = 0.46 P = 0.062
serum albumin levels among the nephrotic patients. There was,
however, as listed in Table 4, a significant correlation between
dietary protein intake and various components of leucine turn-
over when control and patient data were pooled. Higher protein
intake led to a greater leucine turnover. The effect of energy
intake on leucine kinetics was less obvious. Figure 2 depicts
the significant positive correlation between dietary protein
intake and leucine release from protein degradation (r = 0.722,
P = 0.001).
DiscussionIn the current study, we assessed whole-body protein turn-
over by measuring leucine flux or the movement of leucine in
and out of body compartments. ‘3C leucine was infused con-
tinuously into the free amino acid pool (plasma in this instance)
where, under isotope and substrate steady-state conditions,
dilution of the ‘3C-labeled leucine occurs either by exogenous
intake or endogenous protein breakdown. By measuring this
dilution, i.e. , plasma enrichment, and by quantifying tracer loss
as expired ‘3CO,, we determined protein degradation and
amino acid oxidation rates. Protein synthesis rate was then
estimated indirectly as the difference between whole-body
leucine flux and leucine oxidation rate.
Using this technique, we found no evidence to indicate
augmented protein catabolism in patients with nephrotic syn-
drome. As shown in Table 3, all parameters of leucine kinetics,
including leucine release from endogenous protein degrada-
tion, irreversible oxidation of leucine, and leucine-protein in-
corporation, calculated using plasma ‘3C leucine enrichment,
were not different between the nephrotic patients and the
healthy control subjects. More importantly, data calculated
from plasma ‘3C KIC enrichment showed downregulation of
all parameters of the leucine kinetics. Generally, flux rates
derived from plasma ‘3C leucine and plasma ‘3C KIC enrich-ment change proportionately ( 13). In the present study, leucine
appearance rate into the plasma leucine pool was proportion-
ally higher than its appearance rate into the total body leucine
pool in the nephrotic patients, giving rise to a significantly
higher QICU/QKIC ratio. This is, perhaps, related to the fact that
‘3C leucine, which is normally confined to the plasma com-
partment, may be distributed in a larger volume in the ne-
phrotic patients due to hypoalbuminemia. Such larger volume
of distribution would then give rise to a lower plasma ‘3C
leucine enrichment and a falsely higher leucine appearance rate
into the plasma leucine pool. Because KIC is the intracellular
deamination product of leucine and leucine deamination occurs
intracellularly, measurement of plasma 13C KIC enrichment
after ‘3C leucine infusion gives a more precise quantification
of the true leucine appearance into the total body leucine pool
( I 3, 14). As a result of this belief, greater emphasis is generally
given to the reciprocal pool data. Protein flux rates (Qprotcn)’
derived from plasma ‘3C KIC enrichment, were 3.99 and 4.72
14O�
120�
C0
�0a
II?
100�‘ �a
80�
60
over. As shown in Table 4, all parameters of leucine turnover
are significantly correlated with dietary protein intake; higher
intake accelerated leucine flux. Figure 2 plots the relationship
between leucine release from protein degradation to protein
intake. The r value was 0.722 and the P value was <0.001 ;
in this instance, is 0.52, suggesting that 52% of the variance of
I c*� ‘� leucine turnover activity was accounted for by protein intake.
The four nephrotic patients whose leucine fluxes were mea-
sured twice, 20 d apart, identified as 1 and la, 2 and 2a, 6 and
6a, and 8 and 8a, showed remarkable reproducibility of this
measurement.
In healthy subjects, reduced protein intake predictably de-
0 � creases both leucine release from protein degradation and
0 irreversible oxidation of leucine. Motil and associates mea-
r0722 sured leucine flux in healthy young men ingesting diets con-
_o:oo1 taming 1 .5, 0.6, and 0. 1 g/kg per d of protein and found theirp respective leucine release from protein degradation to be 127,
0 6 ‘ I I � I 12 14 109, and 87 �.tmoLfkg per h. The respective leucine oxidationrates for the three diets from high to low protein were, respec-
tively, 1 8, 22, and 13 �mol/kg per h (1 1). Campbell and
colleagues measured leucine fluxes in older subjects, aged 56
to 80 yr, ingesting diets providing either 0.8 or 1 .6 glkg per d
of protein and found that the low protein diet markedly reduces
both leucine release from protein degradation and leucine
oxidation. Protein synthesis was also reduced, but to a lesser
extent (22). Ooodship et a!. reported that reducing dietary
protein intake from 1 .0 to 0.6 glkg per d led to a decrease in
protein breakdown and leucine oxidation in both healthy sub-
jects and patients with chronic renal insufficiency (23). In
another report examining healthy subjects and end-stage renal
disease patients before and after initiation of peritoneal dialy-
sis, Ooodship and associates found that respective protein
intake was I .I , 0.78, and 0.96 g/kg per d, and the rates of
protein breakdown and amino acid oxidation were reduced
proportionately in descending order to protein consumption
(24). Thus, the response of the nephrotic patients was very
similar to the data obtained in healthy subjects and renal failure
patients receiving lower protein intake.
The studies of Kaysen et a!. in humans and in rats suggest
that a moderately reduced protein intake is more beneficial
than higher protein intake in the nephrotic state because the
former diet leads to lesser proteinuria, reduced albumin catab-
olism, and better maintenance of serum albumin levels and
plasma albumin mass (4,5). Our current study documented that
in the nephrotic patients, a protein intake of 0.84 glkg per d did
not lead to accelerated protein catabolism or negative nitrogen
balance. Thus, the common nephrology practice of modest
protein restriction in nephrotic patients appears to be safe.
Incidentally, the level of protein intake in our patients was
similar to the Recommended Daily Allowances of the Food
and Nutrition Board of the National Research Council, Na-
tional Academy of Sciences (25).
In summary, nephrotic patients ingesting a modestly re-
stricted protein diet were able to maintain a positive nitrogen
balance. Leucine turnover kinetics data did not reveal any
evidence of increased protein catabolism. The reduced rates of
protein degradation, amino acid oxidation, and protein synthe-
1072 Journal of the American Society of Nephrology
Protsin Intake (glkglday)
Figure 2. Linear regression between protein intake and leucine release
from protein degradation in nephrotic patients 0 and healthy subjects
.. Higher protein intake resulted in a greater leucine turnover (r =
0.722, P = 0.001). Four nephrotic patients, 1 and la, 2 and 2a, 6 and6a, and 8 and 8a, whose leucine flux was measured twice, showed
remarkable reproducibility of this measurement.
g/kg per d in the nephrotic patients and control subjects,
respectively (P = 0.007). Normally, protein turnover rate is
severalfold the magnitude of daily protein ingestion (20).
Maroni and colleagues recently reported that protein degrada-
tion and protein synthesis were not different between the
control subjects and the nephrotic patients ingesting a similar
diet; they also found no evidence of increased protein break-
down in nephrotic patients (21).
It should be emphasized that in addition to urinary protein
loss, our patients were also given a modestly reduced protein
diet. Under these double sources of protein deprivation, the
nephrotic patients were able to maintain a mildly positive
nitrogen balance. Moreover, during leucine flux measurement,
protein degradation and amino acid oxidation were downregu-
lated, demonstrating their remarkable adaptation in protein
conservation.
The lack of correlation between urinary protein excretion
and the various leucine flux parameters underscores the com-
plexity in the regulation of protein turnover. This process is not
simply dependent on protein loss, but on many other factors,
including protein and energy ingestion, physical activity, hor-
monal profile, the interorgan conversion of metabolites, and
others. In a similar study of nephrotic patients, Maroni et al.
were able to detect an inverse correlation between proteinuria
and leucine oxidation (21).
Although the differences between the nephrotic patients and
the healthy subjects could be attributed to the urinary protein
loss, the lower protein intake in the nephrotic patients is likely
responsible for the reduced rate of leucine and protein turn-
Leucine Turnover in Nephrotic Patients 1073
sis reflect an appropriate adaptive reaction to the lower protein
intake.
AcknowledgmentsThis study was supported by grants from Baxter Extramural Grant
Program (to Dr. Lim), the Bristol-Myers Squibb Pharmaceutical Re-
search Institute, and National Institutes of Health Grants RR55 (Gen-
eral Clinical Research Center) and RR954 (Mass Spectrometry Re-source Center). We appreciate the leadership of Dr. Melissa Cooper at
Bristol-Myers Squibb, the technical assistance of Jerry Fangman,Richard Berger, and Jan Crowley, and the secretarial help of Cassie
Baxter.
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