Changing gravity and weightlessness effects on vasopressin control systems, with immunochemical and biological assay studie

Moran, W. H., Jr.

English

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NATIONAL AERONAUTICS AND SPACE ADHINISTRATION 
PROGRESS REPORT 
Report Prepared by: Walter H. Moran, Jr., M.D. 
For Period : October 21, 1969, t o  February 15, 1970 
Grant: NGR-@-001-019 
Title : The Effect of Changing Gravi.ty and 
Weightlessness on Vasopressin 
Con tr o l  Sy s ten1 s 
A. LAP6/WU SYSTEN 
The LAPG/WU system was developed by Clyde G. Roby, Jr. i n  order 
t o  sitnpXify Vne computer programing e f fo r t  associated with t h i s  project. 
LAP~/WVU i s  a modified version of the LAPG system descri'oed by M.A. Wilkes 
in  LAP^ HhI\TDBOOK", Technical Report No. 2, Compu.ter Research Laboratory? 
Washington University, St. Louis, Missouri (Nay 1, 1967) . LAPG/Ww i s  dasigned 
t o  take advantage of the additional 1024 words of memory available on the basic 
LIN-8  o r  Pi)P=12 manufactured by the Digital  Equipment Corporation. The ex t r a  
memory has been used t o  add (I) chameter by c?tasac%er deletion during the 
edi tor  mode 57hich f a c i l i t a t e s  the preparation o f  input data as well a s  program 
manuscripts, (2) eight character symbolic nanes of which there can be a 
maxiniurfl of  192, (3)  tape identification, and (4) a date which i s  available 
f o r  l i s t i ngs .  A high speed version of LAP6/T;svLs has been developed in which 
the LAP6/iy?N system resides  on a disk.  
w i th  a t  least 6kK of  disk storage. 
Tnis system requires an 8 K  LmC-8 
This manual was designed t o  function as a supplement o t  the %AP6 
HANDBOOKi1 and assumes t h a t  the user has a copy of it available. A l l  con- 
ventions of LAP6, not specif ical ly  contradicted, a r e  identical i n  the two 
systems. Minor changes i n  PROGOFOP have also been made. 
LAPG/ww became operational in October 1968 and has remained in i ts  
The 
present f o m  since January 1969, 
a t  Washington University concerning an acceptable narne f o r  the system, 
system w i l l  be released once a f i n a l  agreement has been reached. 
copies submitted with t h i s  report  a r e  NASA's preprint  copies 
'de have been i n  contact with $1. A. Clark 
Tlze f ive 
B. OF THE M N I O  ASSAY AND THE ANTIDIURETIC BIOASSAY OF VASOPRESSIN 
I n  May, 1969, a comparative study o f  the hunocherflical and the 
biological assays of vasopressin was begun in collaboration with Dr. Hyron 
Miller of the V. A. Hospital i n  Syracuse, New York. 
f ive steps of which f o u r  have been completed. 
Tnis study consists of 
1, CALIBRATION OF REFERXNCE STANDARDS 
A master reference standard was prepared from U.S.P. Posterior 
P i tu i ta ry  Reference Standard powder. 
contained 100 rnU of vasopressin/ml. a s  well as 0,3723 gm. of  disodium ethyl- 
enediamine t e t r a  acetate,  2.0 gm. of sodium chloride, and 0.285 ml. of g lac ia l  
This master reference standard salution 
https://ntrs.nasa.gov/search.jsp?R=19700027448 2020-03-11T23:34:27+00:00Z
ace t ic  acid per l i t e r .  
and shipped t o  Dr. Miller. 
frozen and subsequently thawed a t  the same t h e  t h a t  Dr. IKUer thawed the 
standard t h a t  ha.d arrived in Syracuse. 
Five aliquots of the master reference standard (never frozen) were 
assayed i n  the ra t  using our current; reference standard, 
aliquot were used. 
standard tha t  had been frozen and then thawed. 
250 ml. of  the master reference standard was frozen 
A t  the same t h e ,  10 m l .  of t h i s  standard was 
Five doses of each 
The same procedure was carried out on the master reference 
Biological (ant idiuret ic)  Assay of 
Master Reference Standard - l O O m U / m l .  
Frozen Non-frozen -
104.168 111 . 552 
103.677 113.900 
98.634 118.835 
98.2412 79 7644 
0.33 (S.D.) mU/ml. 1.56 (S.D.) mU/rnl. 
h u n o a s s a y  of  Master Reference Standard - l.OomV/ml . 
101.0 6.8 (S,D,) mU/ml.  
The accuracy of the master reference standard was established as 
being approxjmately 10OmU/ml .  and was used by both laboratories as a cowon 
reference poin t  i n  the following steps,  
2. RECOVERY cu__ AN4 LINEARITY OF NEX3ODS 
In the second step, calculated cmounts of master reference standard 
The aiiounts of added vaso- were added t o  fresh plasma pr ior  t o  extraction. 
pressin were calculated t o  f a l l  within a range tha t  could. eas i ly  be detected 
by both metnods, 
chromatographed on CG-JO resin columns. 
columns were s-pl5-t and 'c'nen evaporated t o  dryness. 
redissolved i n  r a t  inject ion solution f o r  bioassay. 
dissolved i n  @hosphate buffer (pX 6) ,  frozen and then shipped t o  Syracuse 
for  jmmunoassay. 
The four plasma samples were extracted with TCA and them 
The acid-ethanol eluates froni t h e  
l / 3  of each eluatg was 
The other 2 / 3  was re- 
SAMPLES 
60 ml. of plasma + No vasopressin 
30 ml. of plama + 1500 &U of vasopressin lx 
2x 30 rnl, of plasma + 3000 pU of vasopressin 
3x 30 ml. of plasma -I- 4500 pU of vasopressin 
glx 
The following r e su l t s  were obtained : 
-Z-' , 
2X 2862 100 
2863 100 
2864 100 
2865 100 
2866 100 
5.20 
5.41 
4.56 
4.81 
47.2 
42.3 
41.4 
41.5 
222. 
41.06 2.30 
95 .2 
94.8 
90.9 
91.5 
!zu 
93.62 2 1.00 
153. 
142, 
148. 
154 . 
150.4 & 2.42 
24.9 
%.68 
8.12 
11.26 
55 e 7 2  
60.0 
65.6 
71.88 
5 6.71 
87.6 
123.0 
a4.4 
90J 2 8.33 
78.2 
78.8 
109.4 
104.4 
100 .o 
2 6.58 
The biological assay vas l i nea r  and closely approximated the 
theoretical  concentration of vasopressin ( i f  the losses during ex-trac-Lion 
of plasma taken i n t o  account). 
1W.t of i ts  sens i t iv i ty  F J i t h  the 
of variation of 59%. 
fix, lX, and 2X values. 
different  . 
The immunoassay was w o r k h g  a t  the lower 
sample a s  evidenced by the coefficient 
The 3 value f e l l  f a x  belov Vne l inear  extension of 
The two l i n e r  regressions were not s t a t i s t i c a l l y  
3. SEII1,OUALITATIVE COI?Ip-4RISON 
The third s tep  was more complicated. It consisted of three parts.  
For the first and second parts, 30,000 pU of  master reference standard was 
added t o  50 m l .  o f  whole blood. 20 ml. of this blood sample was centrifuged 
and the plasma was extracted with TCA and chronatographed on CG-JO resin. 
The acid-ethanol eluate w.zs then collected i n  2 nil. f ract ions numbered 2900- 
2909. These were s p l i t  and assayed in the same manner a s  in step 2 ,  The 
values below represent the recovery of 12,000 uU of ADE from 20 ml. of i;rhOle 
blood . 
Sample 
N o t  
2900 
29 01 
2902 
2903 
2905 
29 06 
2907 
2908 
2909 
2904 
ADH Bioassay 
PU 
68,2 
3470 
3170 
278 
53.3 
45,2 
31 *9 
25 .o 
NeD,  
NeD,  
2500 
2219 
N e D t  
MeDe 
N e D t  
N t D e  
N e D t  
MaDe 
N t D e  
The values from the immunoassay indicate  t h a t  the biological and 
islullunochemical a c t i v i t i e s  of vasopressin have the sams coeff ic ient  of 
d i s t r ibu t ion  during adsorption chromatography on CG-50 res in  columns. 
lack  of adequate sens i t i v i ty  prevented the ianmunochemical method from demon- 
s t r a t ing  the en t i r e  curve, 
The 
Another 20 ml. of the whole blood sample t o  ru’hich 12,000 pU of 
vasopressin had been added was processed. 
and processed as a separate sample. 
three times t J i th  sal ine and then laked with d i s t i l l e d  water. Each TCA ex t rac t  
of the plasma sample was saved separately. 
were carried out. 
This t h e  each s tep  was saved 
The red cells were separated, washed 
Tnree additional 58 TCA cxtyactioiis 
TCA-1 - 10% 
TCA-2 - 8% 
TCA-3 - 55 
TCA-4 - 5% 
TCA-5 0 9% 
TCA-6 - 5% 
TCA-7 - 5% 
LAKED RBC’S 
NACL - 1 
NACL - 2 
NACL - 3 
Saflple ADH Bioassay 
No. P* 
2910 
2911 
2912 
2913 
2914 
2915 
2916 
2917 
2918 
2919 
2920 
’ 6040 
1770 
160 
90.2 
4- 
N t D e  
I- 
N t D .  
40.6 
47 . 7 
691 
ADH Imraunoassay 
PU 
5469 
688 
24 
N e D t  
N t D e  
N e D e  
N t D t  
N t D e  
N t D t  
N a D e  
3000 
Again the irmnmochemical a c t i v i t y  Matched the biological a c t i v i t y  of 
vasopressin with the l imitat ion o f  s ens i t i v i ty  of the inmunoassay s t i l l  evident, 
The th i rd  se r i e s  2922-2927 consisted of a r t i f i c a l  plasma with vaso- 
pressin added t o  equal vasopressin concentrations t h a t  would be encountered 
in normal plasma. 
forced t o  work a t  l/3 of  i t s  usual sens i t iv i ty ,  
This p a r t  did not  work out t o o  well as the bioassay w a s  
ADH Added ADH Bioassay ADH Immunoassay Sample 
NO 0 pU/nl* pU/nl l  e f i u / m l  . 
2922 0.6 
2923 0 .o 
2924 1.6 
2925 0.2 
2926 0.0 
2927 2.5 
NOD. 
NeDo 
1.18 
0.46 
?. 
4- 
NOD. 
NOD. 
NOD. 
NOD. 
N.D. 
M.D. 
mesa nornial plasma l eve l s  were beyond the reach of the immunoassay 
even with the CG-50 resin column concentrating procedure. 
4. VASOPmSSIN IN RAT URINE 
Because o f  the lack of an adequate sens i t iv i ty  f o r  the assay of  
plasrna vasopressin levels ,  D r .  Miller tv.rned. t o  the use of the irmunochemical 
assay f o r  the estimation of the vasopressin content o f  urine. In  t h i s  ex- 
periment, f ive  samples of urine collected from normal r a t s  and f ive  samples 
o f  urine collected from r a t s  with her i ta ry  diabetes insipidus were assayed 
in the manner described i n  s tep 2. 
by e i the r  method. 
No vasopressin was found in the D I  urine 
The values from the normal rats are  l i s t e d  below. 
Sample 
No . 
NL - 1 2954 
N L - 2  29 55 
NL " 3 29.56 
NL-4  2957 
N L - 3  29 58 
ADH Bioassay ADH h u n o a s s a y  
r-irr '_ pu 
645.5 2 20.6 (n=4) 480 
382.5 f 18.5 (n=4) 420 
944.7 5 53.4 (n=4) 950 
1298. h 71.9 (n=4) 1250 
919.0 2 58.9 (n=4) 9x0 
Again, a f a i r l y  good correlation was obtained. 
I think tha t  the r e su l t s  obtained so  fa r  indicate t h a t  the jnununo- 
chemical assay o f  vasopressin a s  perfomed i n  Dr. >I.Iiller's laboratory 
accurately deternines the vasopressin content of urine. 
materials a re  not  sciisitive enough t o  be used in determining ADH in hwnan 
plasma e 
H i s  present 
The f ina l  s tep w i l l  involve a sophisticated chromatographic t e s t  of 
specif ic i ty  and i s  i n  the process of being performed. 
c . PUBLIC&4TIONS 
1. Moran, V.H., Jr.: The endocrine response t o  injury, Journal of 
S t  . Barnabas Pledical Center, m: 2-9 (1970) . 
AF'F'ENDIX I - PhGE 1 
E 19 
E 20 
E 21 
E 22 
E 23 
E 31 
E 32 
E 33 
E 3 4  
E 39 
E 48 
E 41 
E 42 
E 43 
E 44 
E 45 
E 46 
E 4 7  
E 43 
E 5c1 
E 53 
E 54 
E 5 5  
E 57  
E 58 
E 61 
E 63 
E 6 4  
E 6 5  
E 66 
E 67 
E 68  
E 6 9  
E 7 @ , .  
E 7 1  
E 72 
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E 7 4  
E 75 
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D 7BC4I 
D 80410 
E 7 6  
E 77  
E 7 8  
A D 3  - S T A B I L I T Y  11 E F F E C T  OF TEMPERATURE 
E F F E C T  OF BLEEO 0:G A D A  A N 9  CRF, 
ADH S T A B I L I T Y  111 REPEATED FREEZE-TdAW PLASMA 
A D H  S T A B I L I T Y  I I I ,  REPZATED FREELE -TH4id STANDARD SO:-UTIO:\l 
A N T I D I U R E T I C  A C T I V I T Y  OF P.NGIOTE;\jSIN 
A:\IGIOTENSif\1 KECOVEKU' 03 CG- 5Gl 3:ESIN COLIJMNS 
AD;I S T A B I L l T Y  V J  N . 4  OXAL4TE AN9 EDTA A S  I ! q H I B I T O : < S  
AD!i RECOVERY 1 1  N O  ETYER KAS;I  
UDt i  S T A B I L I T Y  VI,, NA OXALATE AND E D i . 4  A S  I N $ I G I T O K S  
C Z F  I S O L A T I O N  1, TO AMES 
C R F  I S O L A T I O N  111 TO AXES 
A D d  R E C O V E R Y  I I t  NO E T H E 2  WASH 
ADt-1 R E C 9 V E R Y  111, ?tt 4.0 R E S I N  COLUPlN 
ADif RECOVEXY IV, EE VS N E 5  
ADH R E C O V E R Y  V J  EE V S  NEE 
A D d  RECOVERY V I ,  EE VS NEE 
C.3F I S a L A T I Q N  111, TO A N E S  
AD!4 S T A B I L I T Y  I V ,  EFFECT OF TENPERATLJRE 
VEIThJAM SAMPLES, Ci?F TO AiviES 
A D 3  ISOLATIO!\I 1, D i t i R E T I C  PEAt< 
ADH I S O L A T I O N  11, NAOX A N D  EOTA 0i.q D I U R E T I C  PEAK 
A D 3  RECOVERY V I I ,  PLAS%A 
ADi4 I S O L A T I O N  1x1, N A  I O N  I N  EXTRPCT 
A D 9  ISOLATION i V 3  T d I O G L Y C 9 L A T E  C O R 0  BLOOD 
ADH I s o L A - r f o N  v, T H I O G L Y C O L A T E  COKD ELOOD 
ADH I s L o A T i O i \ i  v r ,  P I T R E S S I N  VS. USP POST a + ~ ~  K E F  I N  mr 
A D H  S T A S I L I T Y  V I I J  STOFiAGE OF DILUTED EXTRACT I N J E C T  SO!_. 
AD3 C H K O ? @ i A T O G 3 9 P i 3 Y  I ,  P I T R E S S I N  
A D 3  C!- tROXATOGRAPH'i  113 E L . U T I O N  FROM PAPEH 
AD3 Ci-iROMATOGKAPdY 111, US? P I T  REF 
ADH CHROMATOGR4PHY I V J  R E S I N  f PAPER i.iSTH USP i<EF 
ANGI OTENS I N CHROYlATOGRAPflY I , 
OAYTOCIN CklF;O,lATOGRAPYY I ,  
A D H  CHROM4TOGRAPdY VI I I ,  PLASMA-LUNG CA POOi-: GM 
CRF ISOLATiOi '4  I U J  TC) AMES 
A D i l  CtiKO?4ATOGf<A?!-tY V J  C O K D  B L O J D  
A D q  RECOVEKY V I  I I >  R E S I N  P L U S  PAPER C H H O N A T O G R A M  
A D f i  CHROMATOGRAPHY V I ,  USF; KEF S T D  - D I S T  CO!:F 
ADH CHROMATOGR4P4Y VI I ,  C G - 5 0  I O J  EXCHANGE COL 
B I D L O G I C A L  H A L F - L I F E  O F  AD!4 I ,  DC)G 
BiClLOGICAL k I A L F - L I F E  OF A D 3  1 1 ,  DOG 
E F F E C T  O F  T I L T  Oiq P.DH S S C R E T I O N  
A D t i  CL-LROI'4ATOGIIAPiiY IX, PLASXA-LUNG CA POOL: !;X 
A D H  CHROMATOGt?APdY X J  PLUSP?A-LUNG CA PCIifL: 'VL'C 
A D H  C H K O M A T O G K A P d Y  X I  J T I  SSUE-NCIRMAL LU?JG: CN 
A P P E N D I X  I - P A G E  2 
E X P E R I N E i ' J T S  SUPPORTED I N  P A R T  BY N G K - 0 0 1 - 0 1 9  
TYFIOUGi-1 3 2 / 1 5 1 7 8  
E 7 9  ADki C K R O M A T O G K A P H Y  .XI 1, T I S S U E - L U N G  CA: iii'd 
E 80 AD3 C H R O M A T O G R A P H Y  X I  11, T I S S U E - L I V E R  M E T A S T -  C N  
H 191933  A D t l  CHROMATOGKAPHY X I V ,  P L A S M A - Y E A D  TRAUMA:  F J  
E 81 AD14 RESPONSE I ,  U R A T E ,  CbtO?f-f, C O S r i  VS. D O S E  
E 82 A P O L L O  9 PLASIyiA SANPL-FS 
E 83 A D H  I M ~ U ~ I O A S S A Y - B I O A S S A Y  C O M P 4 R I S O Q  f 
E 8 4  A DY I M?i UN I 0 A S  S A Y  - El I 0 A S S A Y  COM P AR I S!;:'V I I 
D 7082 B I O L O G I C A L  H A L F - L I F E  OF ADH 111, D O G  
E 85 A D H  I M i ~ U ~ L O A S S A Y - B I O A S S A Y  COf/ i?Ai?ISON I I I 
E 86 S H O C K  PLASi"IA ULTRAFILTRATE I 
D 70613 B I C J L O G I C 4 L  H A L F - L I F E  O r  R D H  I V J  DO!..; 
E 88.  ADH IMMUN IOASSfiY- 'E:  I OASSAY C O X P A R  I S3Sl I V 
E 9 0  A D d  PLASMA, H E A D  T R A U X A  I 
E87 A D H  PLASMA, D I A B E T E S  r x s I m ~ i i s  
E89 A DH PL A S t4 A 9 I i\3 4. P P Z O  2 R I A T E S E C R %. T I 0 N S Y N D R O E 


The effect of changing gravity and weightlessness on vasopressin control systems Progress report, 21 Oct. 1969 - 15 Feb. 1970

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