Urinary Sulfate can Predict Changes in Bone
Resorption During Space Flight
1
Zwart ,
2
Heer ,
3
Shackelford ,
3
Smith
Sara R.
Martina
Linda C.
and Scott M.
1Universities Space Research Association, 2University of Bonn, Germany, 3NASA JSC, Houston, TX
Abstract
Subjects (n=14, 10 male and 4 female) were astronauts on International Space Station Expeditions 22-40 (missions of
124 – 193 d duration, flown between 2009 and 2014). Subjects were 48 ± 6 y at the time of launch. Their average flight
duration was 159 ± 20 d. The protocol was reviewed and approved by the NASA Johnson Space Center Institutional
Review Board, and the Japanese Aerospace Exploration Agency and the European Space Agency Medical Boards.
Written informed consent was obtained from all subjects before they participated in the study.
Urinary sulfate was positively associated with urinary NTX excretion for
both high and low APro:K menus.
The crewmembers were asked to consume prescribed menus for 4-d sessions with either a low (0.3-0.6 g/mEq) or high
(1.0-1.3 g/mEq) ratio of animal protein to potassium (APro:K) before and during flight (two 4-d sessions preflight and four
4-d sessions during flight). The menus were alternated, in a randomized crossover fashion, such that there was one high
and one low APro:K session before flight and two of each menu during flight. The high and low APro:K menus provided
similar intakes of total energy (based on WHO requirements), total protein, sodium, magnesium, phosphorus, and
calcium. A research dietitian met with the crews before the controlled dietary sessions to plan menus.
24-h urine samples and fasting blood samples were collected at the end of the 4-d prescribed diet sessions. Urine
samples were analyzed for collagen crosslinks, including n-Telopeptide (NTX), C-telopeptide (CTX), and helical peptide
(HP) using commercially available kits (Osteomark, Ostex International, Seattle, WA; Osteometer BioTech, Herley,
Denmark; and Quidel, San Diego, CA, respectively). Urinary sulfate was measured using ion chromatography as
previously described (8). Urinary pH was measured in 24-h urine collections using a standard pH meter.
Urinary pH was negatively associated with NTX excretion during flight but
not before flight.
Mixed-effects linear regression modeling setting 2-tailed α to reject the null hypothesis at 0.05 was used to evaluate the
effects of diet and flight on markers of bone resorption (Stata IC software version 12.1, StataCorp, College Station, TX).
Data presented are mean ± SD.
There is a significant amount of variability in the data. While we continue
to evaluate the data set (pending complete data sets for all 17 subjects),
there are many potential factors. Dietary factors such as total energy,
protein (% of kcal), and sodium are being considered, along with other
factors, including atmospheric CO2, exercise, inflammation, and gender.
In this study, we sought to determine whether altering the ratio of dietary animal protein to
potassium affected markers of bone resorption during space flight.
There appears to be more factors during flight that affect urinary pH, which
in turn affects bone resorption.
A high ratio of dietary animal protein to potassium results in a larger
percent change in bone resorption markers and calcium excretion
compared to preflight, but raw NTX values were lower when high dietary
APro:K diets were consumed. Urinary calcium was higher when high
dietary APro:K diets were consumed.
P re flig h t
8
In flig h t
40
7
30
20
10
H ig h A p r o /K
7
U r in e p H
U r in a r y p H
6
References
6
5
5
4
4
1. Smith SM, Heer MA, Shackelford L, Sibonga JD, Ploutz-Snyder L, Zwart SR. Benefits for bone from
resistance exercise and nutrition in long-duration spaceflight: evidence from biochemistry and
densitometry. J Bone Miner Res. 2012;27:1896-906.
L o w A p ro /K
Low
Figure 4. The percent change in urinary ntelopeptide (NTX) excretion during flight (FD, flight
day) when crewmembers consumed a diet with a
higher ratio of animal protein:potassium (Apro/K).
P r e f lig h t
In f lig h t
Figure 5. Urinary pH before and during flight when
crewmembers consumed diets with a high or low
ratio of animal protein:potassium.
0
H ig h
fl
D
F
D
F
Low
1
8
0
1
2
6
D
F
H ig h
0
8
5 .2 5
1
0
4. Frassetto LA, Todd KM, Morris RC, Jr., Sebastian A. Estimation of net endogenous noncarbonic acid
production in humans from diet potassium and protein contents. Am J Clin Nutr. 1998;68(3):576-83.
7. Zwart SR, Davis-Street JE, Paddon-Jones D, Ferrando AA, Wolfe RR, Smith SM. Amino acid
supplementation alters bone metabolism during simulated weightlessness. J Appl Physiol.
2005;99(1):134-40.
D
5 .5 0
0
25
0
5 .7 5
5
50
3. Lemann J, Jr., Relman AS. The relation of sulfur metabolism to acid-base balance and electrolyte
excretion: the effects of DL-methionine in normal man. J Clin Invest. 1959;38:2215-23.
5
F
6 .0 0
0
75
6 .2 5
2. Smith SM, Zwart SR, Heer M, Hudson EK, Shackelford L, Morgan JL. Men and women in space: bone
loss and kidney stone risk after long-duration spaceflight. J Bone Miner Res. 2014;29(7):1639-45.
6. Zwart SR, Hargens AR, Lee SM, Macias BR, Watenpaugh DE, Tse K, et al. Lower body negative
pressure treadmill exercise as a countermeasure for bed rest-induced bone loss in female identical
twins. Bone. 2007;40(2):529-37.
2
100
6 .5 0
10
1
125
6 .7 5
5
5. Zwart SR, Hargens AR, Smith SM. The ratio of animal protein intake to potassium intake is a predictor
of bone resorption in space flight analogues and in ambulatory subjects. Am J Clin Nutr.
2004;80(4):1058-65.
15
D
U r in a r y p H
150
Figure 3. Urinary pH is negatively associated
with atmospheric CO2 concentration during
flight on the International Space Station
(p<0.05).
F
7 .0 0
4
A tm o s p h e r ic C O 2 ( m m H g )
0
L o w A p ro /K
3
6
175
2
t
7 .2 5
1
1
h
H ig h A p r o /K
D
0
Figure 2. Urinary pH is negatively associated with
urinary n-telopeptide (NTX) excretion during flight
(p<0.001), but not before flight.
200
F
2000
N T X n m o l/d
Figure 1. Urinary sulfate positively associated with
24-h urinary n-telopeptide (NTX) excretion during
flight when either a high or low ratio of dietary animal
protein:potassium (Apro/K) was consumed (p<0.01).
re
1500
D
N T X n m o l/d
1000
F
500
5
0
1
1500
D
1000
ig
500
re
0
F
0
P
U r in r a y s u lf a te (m m o l/d )
All subjects and samples have landed and post-flight data collections and
sample analyses are pending for the last 3 subjects.
8
P
In bed rest, an analog of space flight, we showed that the ratio of dietary animal protein to
potassium was associated with excretion of both calcium and collagen crosslinks (5, 6).
This observation was clear after 2-3 weeks of bed rest, but it was not observed in the
same subjects before bed rest. This suggests that the impact of diet on bone is more
pronounced in individuals whose bone is already in a resorptive state. In a separate study
where bed rest subjects were supplemented with essential amino acids including
methionine, urine pH was lower and markers of bone resorption were higher in subjects
receiving the supplement (7).
50
N T X ( % ∆ f r o m p r e flig h t)
Dietary components can influence endogenous acid production due to the presence of
acid and base precursors. Endogenous acids include sulfuric acid produced from sulfurcontaining proteins and amino acids. The quantity of sulfuric acid generated is reflected in
the amount of urinary sulfate excretion (3). Generally, foods containing animal protein are
higher in sulfur-containing amino acids than plant protein sources. Phosphoric acid is
another non-volatile acid that can either be ingested or produced endogenously. Anions,
including conjugate bases of organic acids, make up the majority of dietary base
precursors that the body metabolizes to bicarbonate. Potassium is the predominant
intracellular inorganic cation that balances the charge of organic anions; therefore, dietary
potassium intake can be used to estimate the content of base precursors in the diet.
Frassetto and colleagues developed a model for estimating net endogenous acid
production based on the acid and base precursors in the diet (4). According to this model,
renal net acid excretion can be predicted from two dietary components: total protein and
potassium.
Crewmembers consuming a high ratio of dietary animal protein to
potassium had lower urinary pH both before flight and during flight.
Results
Background
While bone mineral loss associated with space flight is likely predominantly triggered by
the lack of weight bearing, the nature of this loss is multifactorial. Resistive exercise can
mitigate decrements in bone mineral density observed after flight (1, 2), and it is likely that
diet can be optimized to further protect bone. It is clear from the scientific literature that a
suboptimal diet can exacerbate bone loss in healthy subjects.
Summary
U r in a r y c a lc iu m ( m m o l/d )
Mitigating bone loss is critical for space exploration, and diet can play a major role in this
effort. Previous studies showed that dietary composition could influence bone resorption
during bed rest. In this study we examined the role of dietary intake patterns in bone
mineral loss in astronauts during space flight. Crewmembers were asked to consume, for
4 days at a time, prescribed menus with either a low (0.3-0.6 g/mEq) or high (1.0-1.3
g/mEq) ratio of animal protein to potassium (APro:K). Menus were developed for each
crewmember, and were designed to meet both crew preferences and study constraints.
Intakes of energy, total protein, calcium, and sodium were held within ± 5% between the 2
diets. The order of the menus was randomized, and crews completed each set (low and
high) once before and twice during space flight, for a total of 6 controlled diet sessions.
One in-flight session monitored typical dietary intake. On the last day of each of the
sessions, 24-h urine samples were collected, along with a fasting blood sample on the
morning of the 5th day. We report here preliminary data from 14 crewmembers. Three
additional crewmembers’ in-flight samples recently returned from the International Space
Station via SpaceX Dragon capsule. Preliminary analyses show that urinary excretion of
sulfate (normalized to lean body mass) is a significant predictor of bone resorption,
estimated by urinary n-telopeptide (NTX). Dietary sulfate (normalized to lean body mass)
is also a significant predictor of urinary NTX. The results from this study will be important
to better understand diet and bone interrelationships during space flight as well as on
Earth. Funded by the Human Health Countermeasures Element of the NASA Human
Research Program.
Methods
Figure 6. Urinary calcium excretion is higher
when crewmembers consume a high ratio of
dietary animal protein:potassium (p<0.05).
8. Morgan JL, Heer M, Hargens AR, Macias BR, Hudson EK, Shackelford LC, et al. Sex-specific
responses of bone metabolism and renal stone risk during bed rest. Physiological reports. 2014;2(8).