NASA Technical Reports Server (NTRS) 19990052725: An Evaluation of Collagen Metabolism in Non Human Primates Associated with the Bion 11 Space Program-Markers of Urinary Collagen Turnover and Muscle Connective Tissue

37 Pages·0.78 MB·English

by  NASA Technical Reports Server (NTRS)

SUMMARY OF RESEARCH
GRANT NUMBER: NAG-2-1089
GRANT TITLE: An Evaluation of Collagen Metabolism in Non Human Primates
Associated with the Bion 11 Space Program-Markers of Urinary
Collagen Turnover and Muscle Connective Tissue.
PRINCIPAL INVESTIGATOR: Arthur C. Vailas, Ph.D.
Vice Chancellor for Research and Intellectual Property
Management, UH Systems, Vice President for Research,
University of Houston
Co-Director, Connective Tissue Research Laboratory
Tel.'713-743-9104
Fax:[phone]
Email: availas @uh.edu
INSTITUTION: UNIVERSITY OF HOUSTON
4800 Calhoun Blvd
Houston, Texas 77204-2162
PERIOD OF REPORT: 07/01/96 TO02/28/99
I
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AN EVALUATION OF COLLAGEN METABOLISM IN NON
HUMAN PRIMATES ASSOCIATED WITH THE BION 11 SPACE
PROGRAM-MARKERS OF URINARY COLLAGEN TURNOVER AND
MUSCLE CONNECTIVE TISSUE.
Principal Investigator: Arthur C. Vailas, Ph.D.
Vice Chancellor for Research and Intellectual Property Management,
UH Systems, Vice President for Research, University of Houston
Co-Director, Connective Tissue Research Laboratory
4800 Calhoun Blvd
E. Cullen Building
University of Houston
Houston, Texas 77204-2162
Te1:713-743-9104
Fax:[phone]
Email: availas @uh.edu
Co-Principal Investigator: Daniel A. Martinez, Ph.D.
Research Assistant Professor, Department of Biochemistry and
Biology, Division of Molecular and Cellular Biology
Co-Director, Connective Tissue Research Laboratory
University of Houston
Houston, Texas 77204-5513
Tel: [phone]
Fax: [phone]
Email: [email protected]
Technical Monitor: Richard E. Grindeland, Ph.D.
Chief Scientist Bion Program
NASA-Ames Research Center
Moffett Field, CA 94035-1000
Tel: [phone]
NASA-Ames Grant Officer: Beatrice Morales
University Affairs Grants Office
NASA-Ames Research Center
Branch 241-1
Moffett Field, CA 94035-1000
Tel: [phone]
UH-Research Admin.: Barbara DeHaven, Interim Director
Office of Sponsored Programs
Rm 316, E. Cullen Building
University of Houston
Houston, Texas 77204-2 I62
Tel: [phone]
Fax: [phone]
BION 11 FINAL REPORT
UNIVERSITY OF HOUSTON
CONNECTIVE TISSUE
PHYSIOLOGY LABORTORY
TABLE OF CONTENTS
INTRODUCTION ....................................................................................................... 5
METHODS............................................................................................................ 5
RESULTS ............................................................................................................. 6
SUMMARYFINDINGS ................................................................................................ 7
TABLES............................................................................................................... 9
Pre-Flight and Post-Flight Urine BiomarkerAnalyses (Normalized to Creatinine) .................... 10
Pre-Flight and Post-Flight Urine BiomarkerAnalyses (Non-normalized to Creatinine) ............... 11
Urinary Calcium and Osteocalcin Pre-Flight and Post-Flight Analyses ................................. 12
Soleus Pre-Flight and Post-Flight Biopsies Muscle Connective Tissue Analyses ..................... 13
Tibialis Anterior Pre-Flight and Post-Flight Biopsies Muscle Connective Tissue Analyses .......... 14
Medial Gastrocnemius Pre-Flight and Post-Flight Biopsies Muscle Connective Tissue Analyses... 15
FIGURES ............................................................................................................. 16
Urinary Hyp and Hyp/Cr Pre-Flight & Post-Flight Values ............................................... 17
Urinary HiP and HP/Cr Pre-Flight & Post-Hight Values ................................................. 18
Urinary LP and LP/Cr Pre-Flight & Post-Hight Values .................................................. 19
Urinary Total Collagen Cross-links Pre-Flight and Post-Flight Values ................................. 20
Urinary Creatinine Pre-Flight and Post-Flight Values ..................................................... 21
Monthly Hyp Values .......................................................................................... 22
Monthly Hyp/Cr Values ....................................................................................... 23
Monthly I-[P Values ............................................................................................ 24
Monthly HP/Cr Values ........................................................................................ 25
Monthly LP Values ............................................................................................ 26
Monthly LP/Cr Values ........................................................................................ 27
Monthly Total Cross-links (HP+LP/Cr) Values ........................................................... 28
Monthly Creatinine Values .................................................................................... 29
Daily Hyp Values .............................................................................................. 30
Daily Hyp/Cr Values .......................................................................................... 31
Daily HP Values ............................................................................................... 32
Daily HP/Cr Values ............................................................................................ 33
Daily LP Values ................................................................................................ 34
Daily LP/Cr Values ............................................................................................ 35
Daily Total Cross-links (HP+LP/Cr) Values ............................................................... 36
Daily Creatinine Values ....................................................................................... 37
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INTRODUCTION
Patientsexhibitingchangesinconnectivetissueandbonemetabolismalsoshowchangesin urinary
by-productsof tissuemetabolism.Furthermore,thechangesin urinaryconnectivetissueandbone
metabolitesprecedealterationsatthetissuemacromoleculalrevel. AstronautsandCosmonautshave
alsoshownsuggestiveincreasesin urinary by-productsof mineralizedandnon-mineralizedtissue
degradation.Thus,theideaofassessingconnectivetissueandboneresponsein spaceflightmonkeys
by measuremenotf biomarkersin urine hasmerit. Otherinvestigationsof boneandconnective
histology,cytologyandchemistryin theBion 11monkeyswill allow for furthervalidationof the
relationship of urinary biomarkers and tissue response. In future flights the non-invasive procedure of
urinary analysis may be useful in early detection of changes in these tissues. Purpose: The purpose of
this grant investigation was to evaluate mineralized and non-mineralized connective tissue responses of
non-human primates to microgravity by the non-invasive analysis of urinary biomarkers. Secondly,
we also wanted to assess muscle connective tissue adaptive changes in three weight-bearing skeletal
muscles: the soleus, medial gastrocnemius and tibialis anterior by obtaining pre-flight and post-flight
small biopsy specimens in collaboration with Dr. V. Reggie Edgerton's laboratory at the University of
California at Los Angeles.
METHODS
Twenty-four hour samples were obtained intermittently before flight and after flight, before and
after 1 G simulations of flight, and from vivarium monkeys. Urine volumes were measured and
aliquots were frozen for subsequent analysis.
Collagen biomarker concentrations of urinary hydroxyproline (Hyp), hydroxylysylpyridinoline
(HP cross-links), and lysylpyridinoline (LP cross-links) were assayed by reverse-phase high
performance liquid chromatography (RP-HPLC).
Urinary creatinine (Cr) was quantitated using a colorimetric assay to measure potential muscle
rhabdomyolysis and to normalize the connective tissue and bone mineral bio-marker concentrations.
Bonemineralmetabolismwasanalyzedbyassayingpre-andpost-flighturinary calcium (Ca 2+) by
atomic absorption. Urinary osteocalcin (Oc), a biomarker for bone formation was analyzed by
immunoassay with a polyclonal antibody raised against bovine Oc with tracer and standards derived
from purified Macaca mulatta bone Oc.
Skeletal muscle hydroxyproline, an index of collagen concentration, and the mature collagen cross-
links (HP and LP) were measured on micro-biopsy specimens from the pre-flight and post-flight time
periods from all three groups.
Statistics: The urine measurements were analyzed using a two factorial repeated measures analyses
of variance (ANOVA) with the significance set at P<0.05.
RESULTS
Collagen Biomarkers: Summary Tables (Pre-Flight and Post-Flight) and Graphs (Pre-Post,
Monthly and Daily Analyses) can be found in the appropriate sections.
Results indicate a high daily variance in the urine concenlxation of collagen metabolic biomarkers.
The urinary collagen cross-link content in the postflight urines were significantly greater (P<0.05) in
the Flight group compared to the Simulation and Vivarium groups indicating that more mature collagen
had been degraded during the initial postflight recovery period in the Flight group. Creatinine analyses
showed that in all groups urinary levels were significantly elevated (92%-111%) from preflight and the
urinary content of non-reducible collagen cross-links (nM HP+LP/mM Cr) were overall greater during
the postflight period (21%-35%).
Bone metabolism: Urinary Ca z÷ measures were significantly lower (Flight -72%, Simulation
-57%, Vivarium-31%) after flight versus preflight measurements (P<0.05). Oc analyses indicated
that Flight (-43%) and Vivarium (-32%) had significantly lower Oc values postflight.
Muscle Connective Tissue: In the soleus muscle, the only significant changes observed were in the
Vivarium group. The Hyp values were significantly greater post-flight versus preflight values
(P<0.05). There was a trend towards decreased LP values post-flight compared to pre-flight values
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(P<0.09). No significantchangeswerefoundin theFlight or Simulatedsoleusmuscleconnective
measurementsIn. thetibialisanterior,theonlysignificantdifferenceswerefoundin theFlightgroup.
Post-flightHyp wassignificantlysmallerin theFlight groupin contrastto pre-flightmeasurements
(P<0.05).However,HPandTotalCross-linkmeasurement(sHP+LP)tendedtobelargerpost-flight
comparedtopreflightmeasurement(sHP:P<0.07) and (HP+LP: P<0.09) respectively. The medial
gastrocnemius muscle demonstrated no significant differences in any measurements post-flight versus
preflight (P>0.05)
SUMMARY FINDINGS
Biomarker profiles can be used as a first level non-invasive analyses to determine if connective
1,
tissue metabolism has been altered following spaceflight. Initial postflight measurements of
collagen and mineral biomarkers, demonstrated in the first days following flight in the Daily
Graphs, suggest an increase in mineralized and non-mineralized connective tissue turnover in the
Flight group.
Elevated urine Hyp, HP+LP cross-link levels together with reduced urine Ca 2÷levels may indicate
.
new collagen secretion and Ca 2÷ storage, signifying new bone formation during postflight
recovery.
Biomarker analyses investigating the temporal transitions of whole body collagen and mineral
.
metabolism would be greatly enhanced if future experiments facilitated daily in-flight sample
collection of urine from the primates aboard the unmanned space habitat.
Overall, no dramatic muscle connective tissue changes were observed post-flight in the SOL, MG
.
or TA muscles. However, there was a significant decrease in skeletal muscle collagen in the post-
flight TA biopsies in the Flight group with an accompanying increase in muscle collagen cross-
linking suggesting that the interstitial collagen is older "more mature" collagen. This findings were
not observed in the SOL or MG muscle biopsies.
Fourteen days exposure to microgravity may not have been an adequate interval of time to view
.
changes in skeletal muscle connective tissue protein. However, changes at the molecular level of
collagen synthesis (measures of mRNA for COL 1A1, COL 3A1 and Lysyl Oxidase) may be
evident when analyzed.
The biopsy samples received from Dr. Reggie Edgerton's laboratory were smaller in mass than
.
what our current molecular biology methodology could quantitate. If our Laboratory receives
further funding, we will attempt to optimize our mRNA assays for less than -0.5-2.0 mg tissue
wet weight.
TABLES
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