GRANT NUMBER:

SUMMARY OF RESEARCH

NAG-2-1089

GRANT TITLE: An Evaluation of Collagen Metabolism in Non Human PrimatesAssociated with the Bion 11 Space Program-Markers of UrinaryCollagen Turnover and Muscle Connective Tissue.

PRINCIPAL INVESTIGATOR: Arthur C. Vailas, Ph.D.

Vice Chancellor for Research and Intellectual PropertyManagement, UH Systems, Vice President for Research,University of HoustonCo-Director, Connective Tissue Research LaboratoryTe1:713-743-9104Fax:713-743-9125Email: availas@uh.edu

INSTITUTION: UNIVERSITY OF HOUSTON4800 Calhoun Blvd

Houston, Texas 77204-2162

PERIOD OF REPORT: 07/01/96 TO02/28/99

https://ntrs.nasa.gov/search.jsp?R=19990047442 2020-07-02T01:45:09+00:00Z

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:

Co-Principal Investigator:

Technical Monitor:

NASA-Ames Grant Officer:

Arthur C. Vailas, Ph.D.

Vice Chancellor for Research and Intellectual Property Management,UH Systems, Vice President for Research, University of HoustonCo-Director, Connective Tissue Research Laboratory4800 Calhoun Blvd

E. Cullen BuildingUniversity of HoustonHouston, Texas 77204-2162Te1:713-743-9104Fax:713-743-9125Email: availas@uh.edu

Daniel A. Martinez, Ph.D.

Research Assistant Professor, Department of Biochemistry andBiology, Division of Molecular and Cellular BiologyCo-Director, Connective Tissue Research LaboratoryUniversity of HoustonHouston, Texas 77204-5513Tel: 713-743-2662Fax: 713-743-2636Email: ddam@uh.edu

Richard E. Grindeland, Ph.D.

Chief Scientist Bion ProgramNASA-Ames Research Center

Moffett Field, CA 94035- I000Tel: 415-604-5756

Beatrice Morales

University Affairs Grants OfficeNASA-Ames Research CenterBranch 241-1

Moffett Field, CA 94035-1000Tel: 650-604-5811

UH-Research Admin.: Barbara DeHaven, Interim Director

Office of Sponsored ProgramsRm 316, E. Cullen BuildingUniversity of HoustonHouston, Texas 77204-2162Tel: 713-743-9201Fax: 713-743-9227

BION 11 FINAL REPORT

UNIVERSITY OF HOUSTONCONNECTIVE TISSUE

PHYSIOLOGY LABORTORY

3

TABLE OF CONTENTS

INTRODUCTION....................................................................................................... 5

METHODS ............................................................................................................ 5

RESULTS ............................................................................................................. 6

SUMMARY FINDINGS ................................................................................................ 7

TABLES ............................................................................................................... 9

Pre-Flight and Post-Flight Urine BiomarkerAnalyses (Normalized to Creatinine) .................... 10Pre-Flight and Post-Flight Urine BiomarkerAnalyses (Non-normalized to Creatinine) ............... 11Urinary Calcium and Osteocalcin Pre-Flight and Post-Flight Analyses ................................. 12Soleus Pre-Flight and Post-Flight Biopsies Muscle Connective Tissue Analyses ..................... 13Tibialis Anterior Pre-Flight and Post-Flight Biopsies Muscle Connective Tissue Analyses .......... 14Medial Gastrocnemius Pre-Flight and Post-Flight Biopsies Muscle Connective Tissue Analyses... 15

FIGURES ............................................................................................................. 16

Urinary Hyp and Hyp/Cr Pre-Flight & Post-Flight Values ............................................... 17Urinary HP and HP/Cr Pre-Flight & Post-Flight Values ................................................. 18Urinary LP and LP/Cr Pre-Flight & Post-Flight Values .................................................. 19Urinary Total Collagen Cross-links Pre-Flight and Post-Flight Values ................................. 20Urinary Creatinine Pre-Flight and Post-Flight Values ..................................................... 21Monthly Hyp Values .......................................................................................... 22Monthly Hyp/Cr Values ....................................................................................... 23Monthly HP Values ............................................................................................ 24Monthly HP/Cr Values ........................................................................................ 25Monthly LP Values ............................................................................................ 26Monthly LP/Cr Values ........................................................................................ 27Monthly Total Cross-links (HP+LP/Cr) Values ........................................................... 28Monthly Creatinine Values .................................................................................... 29Daily Hyp Values .............................................................................................. 30Daily Hyp/Cr Values .......................................................................................... 31Daily HP Values ............................................................................................... 32Daily HP/Cr Values ............................................................................................ 33Daily LP Values ................................................................................................ 34

Daily LP/Cr Values ............................................................................................ 35Daily Total Cross-links (HP+LP/Cr) Values ............................................................... 36Daily Creatinine Values ....................................................................................... 37

4

INTRODUCTION

Patientsexhibitingchangesin connectivetissueandbonemetabolismalsoshowchangesin urinary

by-productsof tissuemetabolism. Furthermore,the changesin urinary connectivetissueand bone

metabolitesprecedealterationsat thetissuemacromolecularlevel. Astronautsand Cosmonautshave

also shown suggestiveincreasesin urinary by-productsof mineralizedand non-mineralizedtissue

degradation.Thus,the ideaof assessingconnectivetissueandboneresponsein spaceflightmonkeys

by measurementof biomarkersin urine has merit. Other investigationsof bone and connective

histology,cytology andchemistryin the Bion 11 monkeyswill allow for further validationof the

relationshipof urinarybiomarkersandtissueresponse.In futureflights thenon-invasiveprocedureof

urinaryanalysismaybeusefulin earlydetectionof changesin thesetissues.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-flight urinary 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 concentration 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 2÷ 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

6

(P<0.09). No significantchangeswere found in the Flight or Simulatedsoleusmuscleconnective

measurements.In thetibialis anterior,theonly significantdifferenceswerefound in theFlight group.

Post-flightHyp wassignificantlysmallerin the Flight group in contrastto pre-flight measurements

(P<0.05). However,HP andTotal Cross-linkmeasurements(HP+LP) tendedto be largerpost-flight

comparedto preflight measurements(HP: 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

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.

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