Protein metabolism during weight reduction with very-low-

energy diets: evaluation of the independent effects of

protein and carbohydrate on protein sparing13
Jorge A Vazquez, Uzma Kazi, and Navid Madani

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ABSTRACT The aim of this study was to assess the inde- provide greater amounts of carbohydrate ( 75 g/d) and lesser
pendent effects of carbohydrate and protein intakes in protein amounts of protein (30-70 g/d) (3-5).
sparing during weight reduction. Forty-eight obese women were Studies of protein sparing during weight reduction with
randomly assigned to consume isoenergetic (2500 kJ/d) liquid VLEDs in these extremes have shown conflicting results (6-
diets that provided the following amounts (g/d) of protein and 13). For instance, some studies have shown protein sparing
carbohydrate, respectively, for 28 d: 50 and 10, 50 and 76, 70 and with high-protein, low-carbohydrate (5, 6) VLEDs; others have
10, and 70 and 86. The effects of carbohydrate and protein were shown better protein sparing with diets that provide moderate
analyzed by repeated-measures analysis of variance (ANOVA). amounts of protein but high amounts of carbohydrate (7); and
Carbohydrate significantly affected daily urinary ammonia and still others have shown no difference at all (8). Nonetheless, the
total nitrogen excretion, stool nitrogen, and nitrogen balance. Pro- relative importance of the protein and carbohydrate contents in
tein, independently of carbohydrate, significantly affected daily protein sparing is difficult to determine from these studies. For
urinary ammonia, urea, and total nitrogen excretion but had no the most part, previous studies have investigated a single
effect on nitrogen balance. Cumulative nitrogen losses (mmol/28 amount of protein (7-9) or a single amount of carbohydrate
d) were lower in the high-carbohydrate groups than in the low- (11). When more than one amount of protein has been exam-
carbohydrate groups (1869 ± 392 and 361 1 ± 328, P = 0.003) but ined, the VLEDs were not isoenergetic (1 1) and differed in
were similar in the groups receiving 50 and 70 g protein/d (3171 other aspects, including the amount of carbohydrate (6, 8-1 1),
± 327 and 2326 ± 430, respectively, P NS). These results and when more than one amount of carbohydrate has been
indicate that carbohydrate and protein have independent but addi- examined, the VLEDs were not isonitrogenous (6, 8, 10, 1 1).
tive protein-sparing effects during weight reduction. Am J The aim of this study was to assess the independent effects
C/in Nutr 1995;62:93-103. of protein and carbohydrate on protein sparing during treatment
with VLEDs. We studied 48 women in a metabolic wand while
KEY WORDS Obesity, weight reduction, nitrogen bal- they consumed isoenergetic VLEDs that provided two amounts
ance, protein metabolism, very-low-energy diets of protein (50 or 70 g/d) and two amounts of carbohydrate (10
on > 76 g/d). Fifty and 70 g protein/d are considered marginal
and surfeit amounts of protein, respectively, during weight
INTRODUCTION reduction with VLEDs (1-4).

Very-low-energy diets (VLEDs) are frequently used in the

SUBJECTS AND METHODS
treatment of severe obesity (1). These diets are provided either
in liquid or solid form and vary in energy content (1253-3347
Subjects
kJ, or 300-800 kcal/d) and macnonutnient composition (1, 2).
There are many controversies regarding the use of these diets. Forty-eight obese women were studied. Except for obesity,
For example, it is still uncertain whether it is possible to they were in good health as determined by history, physical
achieve zero or positive nitrogen balance at all on these diets, examination, and laboratory evaluation. The study was ap-
whether factors such as sex or body composition influence the proved by the Institutional Review Board of the University of
achievement of nitrogen balance, and what the optimal com- Pittsburgh and all patients gave written informed consent.
position for preservation of lean body mass is (1-3).
I From the Clinical Nutrition Research Unit, Department of Medicine,
The main objective of VLEDs is to produce maximal weight
University of Pittsburgh School of Medicine.
reduction while sparing body protein. However, there is no 2 Supported by grants DK-39157 and RR-00056 from the National
consensus on the optimal VLED composition to achieve this
Institutes of Health. Health Management Resources supplied HMR 70 Plus
goal. In this regard, the major area of disagreement is the
for the study.
relative amounts of protein and carbohydrate needed for pro- 3 Address reprint requests to IA Vazquez, Montetlore University
tein sparing. On the one hand, there are VLEDs that provide Hospital, 200 Lothrop Street, Pittsburgh, PA 15213.
small amounts of carbohydrate ( 30 g/d) and contain mostly Received April 27, 1994.
protein (70-120 g/d); on the other hand, there are VLEDs that Accepted for publication February 27, 1995.

Am J C/in Nutr l995;62:93-103. Printed in USA. © 1995 American Society for Clinical Nutrition 93
94 VAZQUEZ El AL

Experimental design TABLE 1

Daily energy and nutrient intakes from the very-low-energy diets
Patients were housed at the Clinical Research Center of the
(VLEDs)’
University of Pittsburgh Medical Center for 31 consecutive
days. To ensure that all patients began the diet under similar VLED group
metabolic conditions, patients consumed a meat-free, weight-
50P/1OC 50P/76C 70P/IOC 70P/86C
maintenance diet for 3 d (days -3 to -1). This diet provided
75-100 g protein and 10 460-12 552 kJ/d (2500-3000 kcal/d). Energy (Id) 2468 2468 2573 2573
Protein (g) 52.5 50.0 70.5 70.0
On the fourth day (day 0), patients were assigned to consume
Fat (g) 38.0 10.0 32.9 3.0
a VLED that provided 2500 kJ (600 kcal) and 10 g canbohy-
Carbohydrate (g) 10.0 76.0 9.3 86.0
drate/d on to consume an isoenengetic and isonitrogenous
Sodium (mg) 2230 2079 1891 1891
VLED that provided 75 g carbohydnate/d for 28 d (days 0-28). Potassium (mg) 2660 2784 2008 2008
The first 21 patients consumed VLEDs that provided 50 g Calcium (mg) 1984 1904 1044 1400
pnotein/d and the remainder consumed VLEDs that provided 70 Phosphorus (mg) 912 982 1 147 1000

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g pnotein/d. All diets were provided in liquid form. Patients Magnesium (mg) 491 508 442 480
were encouraged to consume nonenergy-containing fluids lib-
, All diets provided vitamins and trace elements to meet or exceed the
erally, and activity was limited to walking in the hospital wand. daily recommended dietary allowances (14). The 5OP/1OC and 50P/76C
Patients were weighed daily in the morning after voiding. very-low-energy diets (VLED5) were prepared by Pfrimmer/Kabi (Erlan-
Temperature, pulse rate, and supine and standing blood pres- gen, Germany), the 70P/1OC VLED was prepared by Food lek (Morris
sunes were measured twice daily. Electrocardiograms were Plain, NI), the 7OP/86C VLED was a modified version of a commercial
performed weekly. Fasting venous blood samples were ob- product, HMR 70 Plus (HMR, Boston). To make the diets similar in energy
tamed at days 0, 14, and 28 of treatment for measurements of and minerals, HMR 70 Plus was supplemented with polycose (25 g/d, Ross
Laboratories, Columbus, OH), NaCI (971 mg/d), and KCI (1 148 mg/d).
plasma 3-hydroxybutyrate (f3-OHB), nonestenified free fatty
SOP, SO g protein/d; bC, 10 g carbohydrate/d; 76C, 76 g carhohydrate/d;
acids (NEFAs), insulin, thynoxin (T4), tniiodothynonine (13),
86C, 86 g carbohydrate/d.
electrolytes, glucose, uric acid, amino acids, albumin, prealbu-
mm, and netinol-binding protein concentrations. At the end of
28 d, the diet was changed to solids that provided 4184 kJ Stool collection and analysis
(1000 kcal) and 100 g pnotein/d, and patients were discharged
Stool was collected in 4-d pools and stored at 4 #{176}C
until they
to continue dieting at home.
were homogenized with distilled water. Samples of the homo-
genate were stoned at -20 #{176}C
until the pools were analyzed for
Diets nitrogen. The results of the analysis were divided by four to
provide an estimate of daily stool nitrogen losses.
The four VLEDs were isoenergetic (2468-2573 kJ/d, on
The nitrogen content of the diets, urine, and stool were
590-615 kcal/d) and provided either 50 or 70 g high-quality
determined by a micro-Kjeldahl technique. j3-OHB was mea-
protein/d. The sources of protein were lactalbumin, milk pro-
sured enzymatically (15). NEFAs were measured enzymati-
tein, casein, and whey. For each amount of protein, there was
cally by using a commercial kit (Wako Chemical USA, Rich-
a low (10 g/d) and a high (76 or 86 g/d) carbohydrate VLED.
mond, VA). Blood urea nitrogen (BUN) and ammonia were
For simplicity, we will refer to these VLEDs as SOP/bC,
measured spectrophotometnically (Sigma Chemical Co, St
50P/76C, 70P/1OC, and 70P/86C, respectively, where P is
Louis). Prealbumin and retinol-binding protein were measured
protein and C is carbohydrate (Table 1). The VLEDs also
by radialimmunodiffusion (Behninger Diagnostic, Somerville,
provided similar amounts of sodium, potassium, calcium, phos-
NJ). Insulin, 14, and 13 were measured by radioimmunoassay
phorus, magnesium, vitamins, and trace elements that met the
(ICN Biomedicals mc, Costa Mesa, CA). Plasma amino acids
recommended dietary allowances (14). All diets were onigi-
were measured by HPLC. Albumin, total protein, glucose, and
nally in powder form and were prepared fresh daily by adding
uric acid were measured at the clinical laboratories of the
distilled water. Compliance was monitored by direct 24-h ob-
University of Pittsburgh Medical Center. Nitrogen balance
servation of patients and by daily measurement of urinary
(mmol/d) was calculated as nitrogen intake minus the sum of
acetoacetic acid concentration (Labstix; Miles Laboratories,
the 24-h urine nitrogen plus stool nitrogen plus insensible
Elkhart, IN).
nitrogen losses (5 mg . kg . d). Because BUN was not
measured daily, nitrogen balance was not corrected by the
Urine collections changes in BUN pool. Lean body mass was measured by
Urine was collected every 24 h and stored without preser- bioelectrical impedance analysis (16).
vatives in plastic containers kept at 4 #{176}C
during the collection.
At the end of each period, the volume was measured and Statistics

aliquots were placed in tubes containing 20 L 2 mol HC1/L or Statistical analyses were done by using segment 4V of the
a few crystals of thymol. The samples were then frozen at BMDP statistical package (17). The data were analyzed by
-20 #{176}C
until analyzed. Menstrual periods occurred during the repeated-measures analysis of variance (ANOVA) using the
studies in a few subjects. To prevent contamination with blood, carbohydrate (10 and > 76 g/d) and protein (50 and 70 g/d)
the urine collections were discontinued for 2-4 d during those intakes as independent variables. When significant (P < 0.05)
episodes. Urine excretion values during the menstruation days interaction effects were found by ANOVA, pair-wise t tests
were extrapolated from the nonmenstruation days by using were used to assess differences between each of the possible
regression analysis. comparisons of interest. The P values from these t tests were
 PROTEIN METABOLISM DURING WEIGHT REDUCFION 95

adjusted upward to control for the multiple comparisons (18). The protein content of the VLEDs did not affect the plasma
The relation of nitrogen balance to plasma hormone concen- -OHB, NEFA, glucose, or creatinine concentrations, but sig-
trations was assessed by regression and correlation analysis. nificantly affected the uric acid and BUN concentrations (Table
The results are presented as mean ± SEM. 3). In the SOP VLED groups, the BUN concentration decreased
from days 0 to 14 and remained depressed at day 28. In
contrast, in the 70P VLED groups, the BUN concentration
RESULTS increased from days 0 to 14 and remained elevated at day 28
(Table 4). These changes were not due to alterations in kidney
Patient characteristics and general effects of diet
function because they occurred independently of changes in
Initially, the groups were similar in age, weight, body mass plasma creatinine concentrations (Table 3).
index (BMI), and lean body mass (Table 2). All four VLEDs Note that in all VLED groups the changes in substrate
were well-tolerated by the patients. Specifically, diarrhea, pos- concentrations were small and the plasma concentrations of all
tural hypotension, gouty attacks, and arnhythmias were not substrates remained within the normal range throughout the

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observed during treatment with any of the VLEDs. The only period of observation.
frequent complaint was constipation. This occurred with equal
frequency in all groups and was treated with stool softeners and Plasma hormones
laxatives. Weight loss was similar in all VLEDs. On average, Hormones are known to influence protein metabolism and to
the mean weight loss after 28 d of treatment was 8.4 ± 0.4 kg, be altered during energy restriction. We therefore measured the
or 7.8 ± 0.2% of initial body weight (Table 2). plasma concentrations of insulin, T4, and 13 at days 0, 14, and
28 of treatment with each of the four VLEDs.
Substrates and hormones The plasma concentrations of insulin and T3 decreased over
The carbohydrate and protein intakes had independent ef- time during treatment with all VLEDs (Table 3). The greater
fects on various plasma substrates. The carbohydrate intake reductions occurred from days 0 to 14. After this time, the
affected the plasma -OHB, glucose, and uric acid concentra- insulin and T3 concentrations remained relatively constant.
tions but had no effect on NEFAs, BUN, and creatinine con- However, the reductions for both hormones were less pro-
centrations (Table 3). The plasma J3-OHB concentration in- nounced in the > 76C than in the IOC VLED groups.
creased over time (P < 0.001) for all VLEDs. By day 14, In comparison with 13, the changes that occurred in 14
f3-OHB had reached a maximum value and stayed at that concentrations over time were modest. Except for the 70P/86C
concentration until the end of the study. However, in the VLED group, the 14 concentrations increased slightly (1.8-
> 76C VLED groups, the -OHB maximal concentration re- 3.0%) from days 0 to 14 followed by reductions at day 28 to
mained within the range observed in obese people after an near day-0 concentrations. 13 was the only hormone that was
overnight fast, whereas the lOC groups exhibited values in the significantly affected by the protein content of the VLEDs
range of ketosis seen after a short fast or high-protein VLED (Table 3).
(Table 4) (19).
Plasma proteins
The plasma glucose concentration decreased over time in all
VLED groups (Tables 3 and 4). However, the reductions from The VLED composition may affect the concentrations of
days 0 to 14 were less pronounced in the > 76C (11-13%) than circulating plasma proteins. Because of their shorter half-lives,
in the 1OC VLED groups (28-38%). the plasma concentrations of retinol-binding protein and pre-
The plasma uric acid concentration increased during treat- albumin are considered to be more sensitive indexes of protein
ment with all VLEDs (Table 3). In all groups, the highest deprivation than are proteins with a longer half-life, such as
concentrations were observed at day 14. The increases were albumin. From days 0 to 14 of treatment, the plasma concen-
less pronounced in the > 76C than in the 1OC VLED groups. trations of retinol-binding protein and prealbumin decreased by
Moreover, by day 28 the values had returned to near or below 24-36% and remained low at day 28 (Table 5). At the same
day-O concentrations in the > 76C VLED groups, but remained times, there were also smaller (2-8%) reductions in the plasma
elevated in the IOC VLED groups (Table 4). albumin and total protein concentrations. The reductions in the

TABLE 2
Initial patient characteristics’

VLE D group

50P/IOC (n 10) 50P/76C (n = 1 1) 70P/1OC (a 14) 7OP/86C (n 13)

Age(y) 44±4 42±4 48±2 44±3

Weight (kg) I 16 ± 6 120 ± 6 103 ± 4 98 ± 4
BMI(kg/m2) 44±2 46±3 38±1 36±1
LBM (kg) 50 ± 2 51 ± 2 47 ± 1 45 ± I
Weight loss (kg) 8.8 ± 0.4 8.9 ± 0.6 8.5 ± 0.3 7.6 ± 0.4
Weight loss (%) 7.5 ± 0.4 7.5 ± 0.4 8.4 ± 0.2 7.8 ± 0.3

‘ S ± SEM. LBM, lean body mass calculated by using bioelectrical impedance analysis; VLED, very-low-energy diet. There were no significant main
effects by ANOVA. See Table I for a full description of the VLEDs. SOP, 50 g protein/d; bC, 10 g carbohydrate/d; 76C, 76 g carbohydrate/d; 86C,
86 g carbohydrate/d.
96 VAZQUEZ El AL

TABLE 3
Plasma substrate and hormone concentrations’

Statistical effects

Group Day 0 Day 14 Day 28 Carbohydrate Protein Time Carb X time Pro X time

/3-Hydroxybutyrate (mol/L) < 0.001 0.994 < 0.001 < 0.001 0.574
SOP/1OC 434 ± 202 2620 ± 400 2849 ± 446
SOP/76C 275 ± 112 735 ± 104 653 ± 84
70P/1OC 390 ± 71 2741 ± 361 3055 ± 404
70P/86C 329 ± 60 666 ± 56 641 ± 45
Free fatty acids (mg/L) 0.291 0.575 < 0.001 0.186 0.923
SOP/1OC 217 ± 16 251 ± 11 287 ± 21
SOP/76C 197 ± 20 263 ± 17 273 ± 33
70P/1OC 189±20 314±24 331±2.8

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70P/86C 240 ± SO 236 ± 23 247 ± 16
Glucose (mmol/L) 0.003 0.184 < 0.001 0.044 0.897
SOP/lOC 5.6 ± 0.3 4.2 ± 0.2 4.1 ± 0.1
50P/76C 5.4 ± 0.3 4.8 ± 0.2 4.6 ± 0.1
70P/1OC 5.8 ± 0.3 4.0 ± 0.2 3.6 ± 0.3
70P/86C 5.4 ± 0.2 4.6 ± 0.2 4.8 ± 0.2
Uric acid (molfL) 0.053 0.033 < 0.001 < 0.001 0.859
50P/1OC 324 ± 35 514 ± SO 454 ± 47
SOP/76C 346 ± 19 382 ± 23 322 ± 27
70P/IOC 242 ± 16 422 ± 46 366 ± 40
70P/86C 304 ± 25 321 ± 29 278 ± 23
Blood urea nitrogen (mmol/L urea) 0.713 0.003 0.002 0.272 < 0.001
SOP/1OC 4.4 ± 0.6 3.6 ± 0.2 3.2 ± 0.2
SOP/76C 4.5 ± 0.4 2.9 ± 0.3 2.7 ± 0.3
70P/1OC 3.9 ± 0.4 4.6 ± 0.2 4.3 ± 0.2
7OP/86C 3.9 ± 0.3 4.8 ± 0.3 4.6 ± 0.4
Creatinine (.tmolIL) 0.395 0.527 0.1036 0.559 0.082
SOP/bC 66±7 78±6 79±7
SOP/76C 71±6 80±4 78±5
70P/bOC 71±2 73±3 79±7
70P/86C 76±4 78±4 71±4
Insulin (pmol/L) 0.061 0.879 < 0.001 0.026 0.464
SOP/1OC 152 ± 26 59 ± 12 82 ± 20
SOP/76C 189 ± 51 111 ± 26 89 ± 13
70P/1OC 149 ± 19 62 ± 9 66 ± 8
70P/86C 170 ± 26 121 ± 19 98 ± 18
Triiodothyronine (nmol/L) 0.003 0.016 < 0.001 0.070 0.194
SOP/1OC 2.0 ± 0.8 1.2 ± 0.1 1.1 ± 0.1
SOP/76C 2.2 ± 0.2 1.7 ± 0.2 1.7 ± 0.2
70P/1OC 1.6 ± 0.1 1.3 ± 0.6 1.0 ± 0.1
70P/86C 1.8 ± 0.1 1.4 ± 0.1 1.4 ± 0.1
Thyroxine (nmoiIL) 0.009 0.940 < 0.001 0.352 0.353
SOP/1OC 89 ± 4 92 ± 3 84±5
SOP/76C 108 ± 9 110 ± 8 107 ± 7
70P/1OC 91 ± 7 96 ± 8 84±8
70P/86C 112 ± S 106 ± 6 99±6

I See Table 1 for a full description of the very-low-energy diets. There were no significant carbohydrate X protein or carbohydrate X protein X time
interactions. SOP, SO g protein/d; 1OC, 10 g carbohydrate/d; 76C, 76 g carbohydrate/d; 86C, 86 g carbohydrate/d.
2 SEM.

plasma protein concentration were similar in all four VLED The most prominent time X diet interactions were those of
groups and were not affected by the carbohydrate or protein alanine and the branched-chain amino acids.
contents of the VLED (Table 5). From days 0 to 14, the concentrations of alanine, phenylal-
anine, and tyrosine were reduced, whereas the concentrations
Plasma amino acids of glutamine, methionine, and threonine were not affected. The
Because plasma amino acids are sensitive indexes of protein concentrations of all these amino acids remained depressed at
metabolism, we measured their concentrations at days 0, 14, day 28.
and 28 of treatment with each VLED. Over time, the concen- Threonine was the only amino acid that demonstrated a
tnations of several amino acids were significantly altered (P < significant carbohydrate effect and methionine was the only
0.01, time effects), whereas others were not affected (Table 5). one that demonstrated a significant protein effect. Threonine
 PROTEIN METABOLISM DURING WEIGHT REDUCTION 97

TABLE 4
Statistically significant interactions, by day’

Day 0 Day 14 Day 28

Carbohydrate x time interactions

-Hydroxybutyrate (pmol/L)
lOC 408 ± 9l 2580 ± 254h 2969 ± 290h

76C 299 ± soa 693 ± 54h.2 630 ± 54I2

Glucose (mmol/L)
lOC 5.4 ± 0.2’ 4.1 ± 01h 3.8 ± 0.2”
76C 5.4 ± 0.la 4.6 ± 0.Ih2 46 ± 0.lh2
Uric acid (mol/L)
1OC 279 ± 17’ 465 ± 32” 408 ± 32C
76C 314 ± 17 346 ± 202 303 ± I9.2

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Insulin (pmol/L)
IOC 144 ± 14’ 58 ± 7” 72 ± 9”
76C 172 ± 24’ 117 ± l5a2 101 ± l4
Alanine (,.mol/L)
IOC 368 ± 23 196 ± 10” 178 ± 10”
76C 327 ± 20 256 ± llh2 251 ± 14h2

Isoleucine (pmol/L)
1OC 60±4’ 92±5” 78±4c
76C s8±3a 76±4” 75±4”
Protein X time interactions
Blood urea nitrogen (mmol/L urea)
SOP 4.4 ± ().9 3.3 ± 05h 3.0 ± 0.5”
70P 3.9 ± 0.4’ 4.6 ± 0.5h2 4.4 ± 0.62

‘ S ± SEM. Means in a row with different letter superscripts are significantly different, P < 0.005. See Table I for a full description of the
very-low-energy diets. bC, 10 g carbohydrate/d; 76C, 76 g carbohydrate/d; SOP, SO g protein/d; 70P, 70 g protein/d.
2 Significantly different from bC, P < 0.005.

was lower in the lOC than in the > 76C VLED group, and whether the observed differences were due to changes in ure-
methionine was lower in the 70P than in the SOP VLED group. agenesis and/or ammoniagenesis, we also measured the excre-
In contrast, the plasma concentrations of leucine and isoleu- tion of these metabolites.
cine increased from days 0 to 14 in all VLED groups. Valine The daily average urea nitrogen excretion was highest during
also increased in all VLED groups except in the 50P/76C the first week of treatment and gradually decreased over time
VLED group. The increases in the branched-chain amino acids in all VLED groups (Table 6). The carbohydrate content of the
were of greater magnitude in the 1OC than in the > 76C VLED VLEDs did not influence urea nitrogen excretion. However, it
groups, but the differences between the groups were small and was significantly affected by the protein content of the VLEDs
not statistically significant. From days 14 to 28, the concentra- (Table 7).
tions of all three branched-chain amino acids decreased, but In contrast with urea nitrogen, there were no time-dependent
they remained higher than the day-0 concentrations in all reductions in the average daily urine ammonia excretion (Table 6).
groups. Nonetheless, the urine ammonia excretion showed significant
carbohydrate and protein effects. Daily urine ammonia excretion
Urine nitrogen was lower in the 76C than in the 1OC groups,
> but higher in the
The largest route of nitrogen loss in the body is the urinary
70P than in the SOP VLED groups (Tables 6 and 7).
tract. To determine whether this route of excretion was differ-
Stool nitrogen
entially affected by the carbohydrate or protein contents of the
VLEDs, we measured total nitrogen daily during treatment. Next, we determined whether the VLEDs affected the losses
The pattern of the urinary excretion was similar with all of nitrogen in the stool. As with urine nitrogen, there was a
VLEDs and showed progressive, significant reductions with reduction in stool nitrogen excretion over time and there was a
time (Figure 1). The total nitrogen excretion was significantly highly significant carbohydrate effect. However, the carbohy-
affected by the carbohydrate content. The daily (Figure 1) and drate effect was opposite to that observed with urinary nitro-
weekly average (Table 6) urine nitrogen excretion was lower gen; ie, the average daily stool nitrogen was higher in the
in the >76C than in the 1OC groups. The carbohydrate effect > 76C than in the 1OC VLED groups (Table 6). There were no
was apparent throughout the 28 d of treatment (Figure 1, Table differences in stool nitrogen between the SOP and the 70P
6). VLED groups.
The urine nitrogen excretion was also affected by the VLED
Nitrogen balance
protein content. The daily (Figure 1) and the weekly daily
average excretions (Tables 6 and 7) were higher in the 70P Nitrogen balance also improved over time in all VLED
groups than in the SOP groups. Urea and ammonia account for groups and was near equilibrium by the fourth week of treat-
most of the nitrogen excreted in the urine of humans. To assess ment in all groups (Figure 1). Nitrogen balance was strongly
98 VAZQUEZ El AL

TABLES
Plasma proteins and amino acids’

Statistical effects
Group Day 0 Day 14 Day 28 Carbohydrate Protein lime Carb X time Pro X time

Retinol-binding protein (mgIL) 0.212 0.898 < 0.001 0.244 0.290

SOP/bC 43±52 31±4 30±4
SOP/76C 46±3 36±3 36±2
70P/1OC 48±2 31±3 29±4
7OP/86C 47±4 36±4 34±3
Prealbumin (mg/L) 0.081 0.853 < 0.001 0.091 0.238
SOP/1OC 218 ± 21 162 ± 17 169 ± 20
SOP/76C 236 ± 17 b9O ± 14 198 ± 10
7OP/1OC 241 ± 22 153 ± 15 142 ± 15
7OP/86C 234 ± 18 196 ± 14 191 ± 19

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Albumin (g/L) 0.238 0.105 0.009 0.773 0.898
SOP/bc 37 ± 1 37±1 36±1
5OP/76C 38 ± 1 39±1 38±1
7OP/1OC 38 ± 1 39±1 38±1
70P/86C 39 ± b 39±1 38±1
Total protein (g/L) 0.457 0.064 0.021 0.993 0.890
SOP/1OC 61±2 60±1 58±1
SOP/76C 63±3 62±3 60±3
7OP/1OC 65±1 66±1 64±2
7OP/86C 68±2 67±2 63±2
Alanine (pmol/L) 0.076 0.521 < 0.001 0.013 0.073
SOP/1OC 340 ± 42 204 ± 20 182 ± 16
SOP/76C 284 ± 22 260 ± 15 268 ± 17
7OP/1OC 383 ± 31 b96 ± 14 175 ± 15
7OP/86C 364±33 248±17 236±21
Glutamine (pmol/L) 0.727 0.123 0.184 0.176 0.533
SOP/bC 485 ± 32 435 ± 30 432 ± 31
SOP/76C 422 ± 21 411 ± 28 420 ± 23
70P/1OC 417±32 389±32 350±28
7OP/86C 400±37 397±30 402±30
Isoleucine (pmol/L) 0.197 0.447 < 0.001 0.020 0.474
5OP/1OC 58±5 99±10 80±7
SOP/76C 65±5 78±6 73±5
7OP/1OC 63±6 87±7 75±5
7OP/86C 53±4 74±6 77±6
Leucine (j.mol/L) 0.225 0.123 < 0.001 0.083 0.719
5OP/1OC 151±11 200±18 172±10
SOP/76C 147 ± 10 166 ± 15 160 ± 13
7OP/bOC 139 ± 16 178 ± 14 144 ± 14
7OP/86C 111 ± 10 154 ± 16 163 ± 16
Methionine (.tmol/L) 0.260 0.039 0.552 0.779 0.415
SOP/IOC 24±3 24±2 26±3
SOP/76C 27±2 24±3 27±2
7OP/1OC 23±2 19±1 18±1
7OP/86C 22±3 24±3 23±3
Phenylalanine (mol/L) 0.329 0.917 0.008 0.084 0.666
SOP/1OC 54±3 48±4 43±3
50P/76C 54±4 49±3 51±5
7OP/1OC 50±4 51±2 44±3
7OP/86C 56±6 50±4 50±5
Threonine (mol/L) 0.030 0.51 1 0.348 0.072 0.244
SOP/1OC 146 ± 19 137 ± 15 147 ± 15
5OP/76C 170 ± 10 176 ± 18 187 ± 15
7OP/1OC 149 ± 12 146 ± 10 140 ± 11
7OP/86C 142±7 172±9 169±12
Tyrosine (mol/L) 0.236 0.828 < 0.001 0.278 0.517
SOP/1OC 71±5 54±4 48±4
SOP/76C 67±4 58±3 54±4
7OP/1OC 62±6 52±5 46±5
7OP/86C 67±11 60±4 59±6
Valine (pmol/L) 0.164 0.853 < 0.001 0.014 < 0.001
SOP/bC 229 ± 22 278 ± 30 236 ± 23
SOP/76C 224 ± 19 216 ± 15 205 ± 15
7OP/1OC 200 ± 21 283 ± 20 230 ± 20
7OP/86C 177±14 230±21 251±22

I See Table 1 for a full description of the very-low-energy diets. There were no significant carbohydrate X protein or carbohydrate X protein X time
interactions. SOP, SO g protein/d; 1OC, 10 g carbohydrate/d; 76C, 76 g carbohydrate/d; 86C, 86 g carbohydrate/d.
2 SEM.
 PROTEIN METABOLISM DURING WEIGHT REDUCTION 99

URINE NITROGEN inverse correlation (r = -0.63, n 30) between nitrogen

balance and serum 13 in the 50P/1OC VLED group, but not in
1200
sop/bc the others (Figure 2). The correlations between nitrogen bal-
-0-- lOP/bC
ance and 14 and insulin plasma concentrations were not sig-
1000 so/isc
7OP/86C
nificant, regardless of how the data were analyzed.

800 Changes in lean body mass

V
0
E To assess changes in lean body mass that occurred during the
E
800
study, we calculated the cumulative (days 0 to 28) nitrogen
losses during treatment with all four VLEDs (Figure 3). The
400 largest losses (3992 ± 351 mmol) occurred in the 50P/IOC
VLED group and the smallest (1723 ± 344 mmol) occurred in
the 70P/86C VLED group. Assuming that 1 kg lean body mass

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,- contains 2355 mmol (33 g) N (20), the average losses of lean
body mass for the 28 d of treatment can be calculated as I .69
NITROGEN BALANCE ± 0.14 and 0.91 ± 0.14 for the SOP/1OC and SOP/76C VLED
groups, respectively, and 1.37 ± 0.23 and 0.73 ± 0.24 for the
80
70P/1OC and 70P/86C VLED groups, respectively. The losses
20
in lean body mass accounted for 9.6-19.2% of the total weight
-40 loss (Table 2).
100 The > 76C VLED groups had significantly lower cumula-
V
tive nitrogen losses than the 1OC VLED groups (1869 ± 392
1S0
E
and 361 1 ± 328 mmol, P = 0.003). The cumulative nitrogen
220 losses were also lower in the 70P than in the SOP VLED
280 groups, but the difference between the two was small and not
statistically significant (2326 ± 430 and 3171 ± 327 mmol,
.34g.
.1. respectively, P 0.279).

0 4 8 1 IS 20 24 28

DAYS
DISCUSSION
FIGURE 1. Urinary nitrogen excretion (top) and nitrogen balance
(bottom) during treatment with the four very-low-energy diets (VLED5). This study shows that various aspects of protein metabolism
i ± SEM. SOP, SO g protein/d; 1OC, 10 g carbohydrate/d; 7OP, during weight reduction are independently affected by the
70 g protein/d; 76C, 76 g carbohydrate/d; 86C, 86 g carbohydrate/d. carbohydrate and the protein contents of the VLEDs. On the
one hand, carbohydrate significantly affects urinary ammonia
and total nitrogen excretion, stool nitrogen excretion, nitrogen
influenced by the carbohydrate content of the VLEDs. The balance (Table 6), and plasma threonine concentrations (Table
effect of carbohydrate was highest during the initial week of 5). On the other hand, protein, significantly and independently
treatment. The daily (Figure 1) and the weekly (Table 6) from carbohydrate, affects urinary ammonia, urea nitrogen,
average nitrogen balances were less negative in the > 76C than total nitrogen excretion (Table 6), plasma BUN (Table 3), and
in the 1OC VLED groups. methionine concentrations (Table 5). The effects of carbohy-
During the first week of treatment, the nitrogen balance tended drate and protein on urinary and stool nitrogen excretion persist
to be less negative in the 70P VLED groups than in the SOP VLED throughout the 28 d of the study but the net effect in nitrogen
groups (Figure 1 and Table 7). As with the carbohydrate effect, the balance was greaten during the initial 14 d of treatment. Never-
effect of protein occurred mainly during the initial week of treat- theless, of the two, the effect of carbohydrate appears to pre-
ment. After this period, the advantage of the 70P groups disap- dominate when both are present in the higher amounts and
peared and, as a result, the overall protein effect was not statisti- becomes the factor that determines the net daily and cumulative
cally significant. Note that the nitrogen balance in the 70P VLED nitrogen losses (Figures 1 and 3).
groups is somewhat overestimated because it was not corrected Carbohydrate exerts its protein-sparing effect via several
for changes in BUN pool (Table 3). mechanisms. One mechanism is by reducing ammoniagenesis
(Table 6). However, this effect is small and unlikely to account
Correlations for the entire action of carbohydrate. Another mechanism is by
To assess hormonal factors that could explain the variations maintaining higher plasma concentrations of hormones such as
in nitrogen balance, we correlated the nitrogen balance data insulin, 13, and 14. These hormones are known to influence
with plasma 13, 14, and insulin concentrations. The data were protein turnover (21, 22) and were all higher in the plasma of
analyzed considering all data points, and separately for each the > 76C than in the 1OC VLED groups (Table 3). These
diet group. When all data points were considered together (n = results agree with previous studies that showed higher plasma
124), there was a significant correlation with 13 (r = -0.23), thyroid hormone concentrations with high-carbohydrate
but not with 14 (r = -0.13) or insulin (r = -0.17) concen- VLEDs (3, 13, 23-25) and with others that have identified 13
trations. However, when the data were analyzed separately for as the most important regulator of protein sparing during en-
each VLED group, we detected a highly significant (P < 0.01) engy deprivation (26).
100 VAZQUEZ El AL

TABLE 6
Nitrogen metabolism’

Sta tistical effects

Carbo- Carb X Pro X

Group Week 1 Week 2 Week 3 Week 4 hydrate Protein Time time time

Urine nitrogen excretion (mmol/d) < 0.001 < 0.001 < 0.0()l 0.848 < 0.O()l
SOP/1OC 742 ± 242 615 ± 17 571 ± 10 538 ± 10
5OP/76C 609 ± 17 517 ± 17 483 ± 14 471 ± 17
70P/bOC 857 ± 30 846 ± 21 819 ± 19 783 ± 15
70P/86C 788 ± 28 766 ± 18 734 ± 20 688 ± 22
Urine urea nitrogen excretion (mmol/d) 0.320 < 0.001 < 0.001 0.830 0.003
SOP/bC 565 ± 34 498 ± 33 451 ± 37 441 ± 22
50P/76C 481 30 426 ± 35 392 ± 35 397 ± 27

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±

7OP/lOC 601 ± 42 648 ± 35 588 ± 43 576 ± 22

7OP/86C 644 ± 49 662 ± 36 546 ± 46 531 ± 47
Urine ammonia excretion (mmol/d) 0.013 < 0.001 0.096 0.208 < 0.001
50P/lOC 25±4 24±3 21±3 27±5
5OP/76C 17±2 14±2 12±2 13±2
7OP/IOC 36 ± 3 4l ± 3 39 ± 3 39 ± 4
70P/86C 35 ± 3 37 ± 2 34 ± 3 32 ± 3
Stool nitrogen (mmol/d) 0.003 0.843 < 0.(X)1 0.108 0.113
SOP/bOC 60 ± 4 50 ± 3 40 ± 2 40 ± 2
5OP/76C 100±5 80±4 70±4 70±4
7OP/1OC SO ± 3 40 ± I 40 ± 4 SO ± 1
70P/86C 70 ± 6 80 ± 6 90 ± 4 60 ± 4
Nitrogen balance (mmol/d) 0.0()3 0.331 < 0.001 0.820 < 0.001
5OP/bOC - 268 ± 27 - 128 ± 17 - 83 ± 11 - 52 ± 11
5OP/76C - 169 ± 19 - 61 ± 16 - 29 ± 14 - 15 ± 16
7OP/1OC - 153 ± 29 - 125 ± 21 - 97 ± 19 - 63 ± 15
7OP/86C -112±29 -81±19 -44±21 +8±23

‘ See Table I for a full description of the very-low-energy diets. SOP, SO g protein/d; bC, 10 g carbohydrate/d; 76C, 76 g carbohydrate/d; 86C, 86 g
carbohydrate/d.
2 SEM.

Our results also provide new insight into the previously correlation between nitrogen balance and serum 13 (or changes
recognized correlation between nitrogen balance and serum 13 in 13) during treatment with VLEDs. We confirmed these
concentrations. Yang and Van Itallie (12), Fislen et al (27), and results, but only in the SOP/1OC VLED group (Figure 2).
Kaptein et al (28) previously reported a significant inverse Collectively, these results suggest that the correlations between
these factors are complex and are affected by the composition
of the VLEDs. For example, Fisler et al (27) and Kaptein et al
5OPI1OC 7OPI1OC
(28) studied patients consuming VLEDs that provided proteins
, - #{149}0
63 , - 121
250
p < 001 p - 0.20

200

150
5000
.
.
. ;. ., .. 1-_. .1!

4500
100
1

0
50 4000

E 0

= E 3500
E

CD 50PI 76C lop!86C 3000

E 250 , -004 , - 0.12

0
00 . p-Oil 2500
CD p - 0.51

200 .
2000
.% 0
150
z 1500
100
.. .%
1000
a
50
E 500
0
0
a n .E 1S 0 100 2t5 4 .o i i us

Nitroaen Balance (mmolldav) Protein intake

FIGURE 2. Relation of plasma triiodothyronine (13) concentration to FIGURE 3. Cumulative (days 0-28) losses of nitrogen during treatment
nitrogen balance for the four very-low-energy diets (VLEDs). SOP, SO g with the four very-low-energy diets (VLEDs). ± SEM. High carbohy-
protein/d; bC, 10 g carbohydrate/d; 7OP, 70 g protein/d; 76C, 76 g drate, VLEDs with > 76 g carbohydrate/d; low carbohydrate, 10 g carbo-
carbohydrate/d; 86C, 86 g carbohydrate/d. hydrate/d.
 PROTEIN METABOLISM DURING WEIGHT REDUCTION 101

TABLE 7
Statistically significant interactions, by week’

Week 1 Week 2 Week 3 Week 4

Urine nitrogen excretion (mmol/d)

SOP 683 ± 23 572 ± iD 532 ± 13C 511 ± 12d

70P 816 ± 2b.2 799 ± l62 768 ± iD2 726 ± 17c2

Urine urea nitrogen (mmol/d)
SOP 526 ± 26 473 ± 20” 442 ± 24” 427 ± 1D
70P 638 ± 28 664 ± 212 570 ± 27 551 ± 32h2
Urine ammonia nitrogen (mmol/d)
SOP 21±2a b8±2a 16±3a 20±3a
7OP 36 ± 2a2 40 ± 22 37 ± 2a2 37 ± 2a2
Stool nitrogen (mmol/d)

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SOP 79±7a 66±D 59±6” 57±7”
70P 62±8a S7±bff’ 64±IP s5±6a
Nitrogen balance (mmol/d)
SOP - 224±21” -101±14”
70P - 129 ± 21.2 99 ± 14” - 66 ± 16’ 20 ± 16d

‘ I ± SEM. Means in a row with different letter superscripts are significantly different, P < 0.005. See Table 1 for a full description of the
very-low-energy diets. SOP, SO g protein/d; 7OP, 70 g protein/d.
2 Significantly different from SOP, P < 0.005.

only. Although Yang and Van Itallie studied three patients groups. This, in part, explains the higher urea, ammonia, and
consuming protein only and three patients consuming 50% of total nitrogen excretions in the 1OC VLED groups compared
energy as proteins and 50% as carbohydrates, the total carbo- with the > 76C groups. We cannot, however, determine
hydrate consumed was less than the amount provided in the whether amino acids from endogenous sources (ie, protein
> 76C VLED used in this study. breakdown) and from dietary proteins are used to the same
An additional mechanism by which carbohydrate may spare degree for gluconeogenesis.
protein is by reducing the rate of gluconeogenesis. After 3 d of The results of this study also show that VLEDs that provide
a complete fast, glycogen stones are depleted and glucose is 70 g pnotein/d are not superior to those providing SO g protein/d
predominately produced de novo (29). Similar events are pre- for protein sparing during weight reduction regardless of
dicted to occur during treatment with VLEDs except that the whether they contain high or low amounts of carbohydrate.
glycogen stores and the rates of gluconeogenesis may vary This conclusion is supported by the following lines of evi-
widely depending on the carbohydrate content of the VLED. dence: First, after the initial week of treatment, there was no
The most important gluconeogenic precursors are lactate, glyc- difference in daily (Figure 1) or weekly (Tables 6 and 7)
erol, and amino acids (30). During a short fast, obese patients nitrogen balances between the 7OP and the SOP VLED groups.
converted an average 55.9% (range 30.4-81.3%) of the glyc- The main mechanism that accounts for the lack of difference is
enol produced by lipolysis into glucose and this accounted for that uneagenesis and ammoniagenesis were higher in the 7OP
8.3% of the total glucose hepatic output (31). Recently, we VLED groups. The origin of the excess ammonia and urea
showed that after an overnight fast in obese women, 46-49% of nitrogen cannot be ascertained from the results of this study,
the glycerol produced from lipolysis was converted into glu- but the most likely source is dietary protein. Second, the
cose and that accounted for 10-11% ofthe total glucose hepatic cumulative losses of nitrogen for the 28 d of treatment are
output (32). During weight reduction with VLEDs, the contni- similar in the 70P and the SOP groups (Figure 3). This is
bution of glycerol to gluconeogenesis increased. However, the surprising considering that we are likely to have overestimated
increase in glycerol rate of appearance and in the rate of nitrogen balance in the 70P groups because we did not correct
conversion into glucose was modest and, at its peak, accounted the results for the changes in the BUN pool (Table 3) and
for no more than 16% of the endogenous glucose production because of the inherent biases of the nitrogen balance at higher
(31, 32). protein intakes (33). Lastly, all other indexes of protein nutri-
To our knowledge, there has been no study of the rate of tion measured such as the plasma concentrations of albumin,
gluconeogenesis from lactate during weight reduction. How- prealbumin, and amino acids (with the exception of methio-
even, because dietary glucose is the major (but not the only) nine) were similarly affected in the SOP and 70P VLED groups.
source of lactate in the body, it is reasonable to assume that Nonetheless, despite these findings, it is prudent to recommend
gluconeogenesis from this precursor will be low during treat- that VLEDs provide 70 g protein/d because this amount
ment with VLEDs, especially when the carbohydrate content is would provide a greater margin of safety for most people.
low. This implies that amino acids are the most important Our results also indicate that the use of > 76 g canbohy-
gluconeogenic precursors during weight reduction with drate/d instead of 10 g carbohydrate/d in an isoenengetic VLED
VLEDs. In this study, patients in the high-carbohydrate VLED will result in the sparing of an additional 0.54-0.64 kg of lean
groups consumed 66-76 g more canbohydrate/d than did the body mass in 28 d, depending on the protein content of the
patients in the low-carbohydrate VLED groups. Everything VLED. We do not know whether the effect of carbohydrate on
else being equal, this would indicate that gluconeogenesis from protein sparing would persist or disappear if patients were
amino acids will be higher in the low-carbohydrate VLED treated for longer periods of time. By 28 d, patients in all
102 VAZQUEZ El AL

groups were in near nitrogen balance (Figure 1), and it is VLEDs that contain low (< 10 g/d) rather than high (> 76 g/d)
reasonable to assume that after that time the differences be- amounts of carbohydrate.
tween the groups would not continue to increase. If that is the In summary, by studying VLEDs that provide two amounts
case, then it can be argued that the relative advantage of of protein and two amounts of carbohydrate, we were able to
high-carbohydrate over low-carbohydrate VLEDs is likely not determine the independent effects of protein and carbohydrate
to be of clinical significance. This is supported by the absence on several indexes of protein nutrition during weight reduction.
of adverse reactions in any of the groups and by the relatively Our results showed that the total carbohydrate and protein
small losses of lean body mass in all VLEDs, which in the intakes were both important for protein sparing during weight
worst group were less than predicted (19.2% of weight loss) reduction. Furthermore, our results showed that these effects
(Figure 3). These results also agree with recent studies of body were additive and the best protein sparing was obtained with
composition after weight reduction with VLEDs (34). There- the VLED that provided 70 g protein and 86 g carbohydrate/d.
fore, it can be concluded that all the VLEDs used in this study Further studies are needed to determine whether greater protein
to a certain extent prevented excessive losses of lean body mass sparing can be achieved with VLEDs that contain higher

Downloaded from www.ajcn.org at Univ of Colorado Hlth Sci Ctr Denison Mem Lib on September 30, 2007
during weight reduction. However, in the interpretation of our amounts of protein and carbohydrate. A
results two factors should be considered. First, our subjects We are indebted to ludy Arch and the nurses of the Clinical Research
were all women, relatively healthy and severely obese. There is Center of the University of Pittsburgh Medical Center for their help during
some evidence to indicate that nitrogen conservation may be the performance of these studies and to Janine Janosky and Kim La for
different in men and women and in moderate and severe statistical assistance.
obesity (35, 36). Therefore, caution should be exercised in
extrapolating these results to other populations. Second, we
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