Changes in amniotic fluid glucose, p-hydroxy-butyrate , glycerol, and lactate concentration in diabetic pregnancy .
A.
ELMORE
LIM M.
!i.
SEEDS,
LEUNG,
WILSON
PAUL, Cincinnati.
T.
M.D.
M.S.
TABOR, RUSSELL,
PH.D. PH.D.
Ohio
Amniotic fluid glucose. POH butyrate, glycerol, and lactate concentrations were measured in 75 samples collected in the third trimester of pregnancy from 50 diabetic patients, all but four of whom required insulin. Increases in maternal fasting plasma sugar were accompanied by corresponding increases in amniotic fluid glucose and on.occasion increases in amniotic fluid pOH butyrate. These data correspond to previous reports of placental glucose transfer and in addition, provide statistically significant evidence of placental @OH butyrate transfer since the hyperglycemic, hyperinsulinemic fetus of a diabetic mother would be a poor primary source for ketogenesis. Relatively poor correlation of elevated fluid levels of these solutes to fetal outcome probably reflects a low incidence of maternal hyperglycemia, ketoacidosis, and over-all reduced neonatal morbidity-mortality rates in this group of metabolically well-controlled, predominantly insulin-requiring diabetic patients managed in a regional high-risk perinataf center. (AM. J. OBSTET. GYNECOL. 135:887, 1979.)
PROPER METABOLIC control of diabetic pregnancy results in maternal blood sugar in the euglycemic range,’ thus preventing increases in plasma ketoacids and glycerol that accompany hyperglycemia in this diseased state. Clinical evidence for proper levels of these solutes is usually obtained by periodic blood sugar measurements and/or determination of urine suger and ketone bodies. It is common in the routine management of the insulin-requiring diabetic pregnancy to perform an amniocentesis late in the third trimester for purposes of assessing fetal maturity. It was felt that perhaps determination of amniotic fluid levels of glucose, glycerol, ketoacids, and lactate could also be helpful in the clinical assessment of the diabetic pregnancy. For example, if elevation of these solutes in maternal
From the Department oJ Obstetrics and Gynecology, Uniuwsity of Cincinnati School of Medicine. Supported in part by United States Public Health Service Grant No. IPOL-HD-11725-01 and by a grant from the Juvrnile Diabetes Foundation. Presented by invitation at the One Hundred and Second Annual Meeting oj the American Gynecological Society, Hot Springs, Virginia, May 16-19, 1979. Reprint request.7:Dr. A. Elmore Seeds, Department d Obstetk-s and Gynrcology, University oJ Cincinnati School of Medzrine, Cincinnat~~, Ohio 45267. 0002-9378/79/230887+09$00.90/O
@I 1979 The C. V. Mosby Co.
circulation is an indication of poor metabolic control or improper management of the diabetic pregnancy and if these solutes cross the placenta in significant quantities to the fetus, it is reasonable to expect that amniotic fluid levels of these compounds might provide an indication of maternal metabolic status in this condition. Except for glucose, there are almost no data on amniotic fluid accumulation of these metabolically significant compounds in diabetic pregnancy. If these solutes cross to the fetus and get into amniotic fluid in significant quantities then depending on their relative ability to escape (i.e., turnover time or disappearance time) they may reflect events in maternal and/or fetal compartments for varying periods of time or function as a memory system. Previous studies have indicated that human placental tissue is readily permeable to glucose and that fetal serum levels parallel maternal levels over a wide concentration range, probably reflecting the facilitated diffusion of this solute.2-J Elevated amniotic fluid glucose concentrations have also been measured in diabetic pregnancies5-” in parallel with maternal hyperglycemia, indicating significant transport of this compound from mother to fetus to amniotic fluid. Comparisons between amniotic Huid glucose levels and perinatal and neonatal outcome have also been re887
888
Seeds et al
Table I. Maternal
fasting plasma glucose and atnniotic SOlUlf.~
Huid solutes for diabetic and nondiabetic
lhhtir
Plasma fasting glurose (mgi 100 ml) Amniotic fluid glucose (mgi100 ml) Lactate (mEq/L) ,kXBH butyrate (mEq/L) (ilyrerol (mgi 100 ml)
119.3 29.6 9.3 0.22 151.9
!Vontliab&
-t 60.8 (60) t 28.5 (7.5) t 3.0 (56) ‘-+ 0.38 (61) f 39.4 (45)
Not 12.5 8.6 0.19 134.4
mea&t,-ed r 6.4 (40) k 2.3 (33) ? 0.23 (25) 2 28.4 (IS)
pregnanc-jes* I’ r’alri? co.0 I ~0.05
-cO,1),5 >o.o:i
*Values given are mrans ? standard deviations. Numbers or observations are in parentheses. Table II. Correlation between paired plastna glucose and amniotic fluid solutes in diabetics* .4mnioticjluid C&mm
Fasting plasma glucose
Amniotic Huid glucose
Lactate @OH hutyrate
0.62!:
WN
Lactatr 0.08 (48) 0.02 (56)
WH
butymte 0.29t (53) 0.55$
(61)
0.04 (55)
Glywrol 0.10 (41) -0.01 (45) 0.03 (41)
-0.08
(44)
*Values given are correlation coefficients, Numbers of observations are compared in parentheses. tCorrelation significant at P < 0.02. fCorrelation significant at P < 0.00 1.
ported and, although interpretations differ, abnormally high amniotic fluid glucose levels appcarecl to correlate with increased perinatal morbidity and mortality rates. These previous studies are preliminary in that they have been performed on small numbers of‘ insulin-requiring diabetic pregnancies, although the incidence of‘ hyperglycemia was surprisingly high. apparently due to relatively inadequatr metabolic control. (:onflicting information from animal studies indicated that ketoacids crossed the placenta in rodents but did not penetrate the ungulate placenta.” There is no clear evidence documenting human placental permeability to these compounds. Increases in fetal ketoacids in the fasting pregnant state have been interpreted as evidence of placental transfer of. these solutes in the Presence of’ very small positi\c human subject.“-‘” umbilical venoarterial differences in POH butyratc concentration and correlations bet\vcen increasing maternal and fetal blood Levels of‘ this metabolite were suggestive hut not conclusi\~e indications of placental transfer. However, when tnaternal and fetal compartments are hypoglycemic and hypoinsulinemic~ and pt-rsumably. then, both ketogenic sources, such increases do not necessarily indiratr placental transfer of ketoacids. No tneasurements have been recorded in the diabetic pregnancy wherein the mother is hyperglycemic arid hypoinsulincmic and the f’etus hyperglycemic with
adequate or increased amounts of iltsulin (thus increased fetal ketoacids ~vo~11d presumahly he of‘ matet.nal origin) to show that elevated ketoacids itt the maternal compartment arc transtnitted across,the placenta to the fetus. Excess lactate will accumuIate in maternal and/or fetal c.otnpartmcnts in response to hypoxia and the possibility exists that increased fetal lactate ma) lead to increased amniotic fluid levels of‘this solute during acute or chronic distress accompanying poor maternal metabolic control of diabetes. Evidence f’w transplacental passage of’ glycerol in animal or man is inconclusive. Thus with the exception of glilpose, little is known about the placental transfer and amniotic fluid accumulation of these solutes which are possibly elevated in the pregnant diabetic \+oman. Therefhrc-, measurements 01’amniotic fluid glucose, P-hvdrt,xybrltyt-air. glycerol, and lactate in diabetic pregnancies were undertaken to determine if these levels wwe altered in thr diabetic pregnancy and, if’ so, did these changes tol-relarr to maternal blood sugar levels and/or fetal ottwmw.
ftllelhodology Sixty amniotic fluid samples were obtained in the late third trimester from 38 pregnant diabetic patients. Maternal fasting blood sugar was measured on the morn ing of each amniocentesis. An additional 15 amniotic fluid satnples were obtained in 12 diabetic patients wherein a maternal fasting blood sugar was not collected. These 50 pregnant diabetic pzatients were distributed according to the White classification as fi)llwvs: Class A, 4; Class B, 14: Class C, 2.5; Class I). 4; Class K, 3. Where sufficient amniotic fluid volume was available, amniotic fluid glucose, P-hydroxybutyratc, glycerol, and lactate concentrations were determined and compared with values collected from 40 nondiabetic control pregnancies at the same gestational age. The p-hydroxybutyriciacetoacetic acid ratio is usually maintained or increased during the rise in ketone bodies accompanying poorly controlled diabetes.‘” Thus dctermination of P-hydroxybutyrate as a valid indicator of ketoacid accumulation in this condition seemed appropriate. Glucose \vas measured en7vmatically l)\
Volume
135
Number
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Changes in amniotic glucose, pOH butyrate, glycerol, and lactate
889
250 -. 8 s $j 0 3
225
.
200 175 150 -
3 125 8 a 100 G” b8 -75 --
.
s
.
T
50 :25 0 0
. :
. -.
. . .z ’ . . .2.
l
. . .;*&. ;; $2 .* * 1 I I I I I I ; 50 loo 150 200 250 300 350 400 FASTING PLASMA GLUCOSE (mg%)
I 450
I 500
CORRELATION Id= 0.62 PcO.001 RANK CORRELATION (r,l=0.70 PCO.001
Fig. 1. Amniotic fluid glucose concentrations are plotted vs. maternal fasting plasma glucose concentrations. The correlation and rank correlation are given for this relationship.
25
50
I I 175 75 100 125 150 AMNIOTIC FLUID GLUCOSE(mg%)
I 200
I 225
I 250
CORRELATION (r)=.55 PO.O5
Fig. 2. Amniotic fluid POH butyrate concentrations are plotted vs amniotic fluid glucose concentrations. The correlation and rank correlation are given for this relationship. means of a glucose oxidase method. Amniotic fluid lactate was measured enzymatically according to the method of Hoehorst.” P-Hydroxybutyrate was measured according to the method of Williamson, Mellanhy, and Krebs. I8 Glycerol was measured as described by Pinter, Hayashi, and Watson.‘g All patients were hospitalized at the University of Cincinnati Medical Center Hospitals for predelivery care and metabolic control at the time of amniocentesis. Amniocenteses were performed from 0 to ‘Ll days prior to delivery. Most of these patients, benefiting from contemporary metabolic management of the diabetic pregnancy, were in reasonably euglycemic range.
Amniotic fluid solute values were correlated with fetal outcome in an attempt to evaluate the prognostic significance of these measurements in the diabetic pregnancy. Neonatal morbidity was defined by the following criteria: Respiratory distress syndrome (RDS)-grunting, tachypnea >60 minute retractions, cyanosis w-ithout added oxygen: all of these signs present for more than 6 hours Respiratory distress-tachypnea: >60/min Hypoglycemia-serum glucose: 530 mgi 100 ml Hypocalcemia-serum calcium: 57 mgi 100 ml for premature infants 58 mg/ 100 ml for term infants
890 Seeds et al.
\
3.0
2 lu g
27 241
it
18-
3&
15-
bF 21c
Q a 3
09-
12-
’ 2 Q
060.3-
$
0.0 0
.
.
** . .
-4 . 22X? :; 1: . : 1.:..I -** 1 . I. I I I loo 150 200 250 300 350 400 50 FASTlNG PLASMA GLUCOSE hq%)
I 450
1 500
CORRELATION (r)=.ZS Ptlae WdL)
Cl00 2100
18.8 2 9.4 (32) 45.0 -1-39.9 (28) p < 0.01
153.2 -1-39.5 (22) 153.8 2 42.5 (19) N.S.
0.15 2 O.OS(25) 0.28 k 0.59 (28) N.S.
9.1 * 2.1 (25) 9.6 AT4.1(23) N.S.
Cl00 100 S 0.05
P-o.05
24.5 k 14.5 (38) 32.5 k 18.5 (7)
>0.05
>0.05
Yes No Yes
9.6 t 4.0 (25) 9.9 T 2.5 (7)
BO.05
>0.05
No
0.13 ? 0.06 (27) 0.51 _’ 0.93 (7)
>0.05
20.05
z-o.05
P-o.05
NO
Amniotic Huid lactate (mEq/L)
fluid solute concentrations
Yes No Yes
143.1 k 48.9(18) 154.7 2 27.8 (6)
891
*Values given are means f standard deviations. Numbers of incidence are in parentheses. Table V. Amniotic
fluid sollute and maternal
Cause of &ath 1. RDS 2. Respiratory disease
fasting plasma glucose values in two cases Amniotic fluid
Maternal ,fa.sting plasma glucose
No. of days separating amniocentesis from deliveq
Glucose (mgllO0 ml)
97
3 20
17.6 25.3
tate, /3-hydroxybutyrate, and glycerol concentrations in the diabetic compared to the nondiabetic group were not statistically significant. However, when paired samples, i.e., plasma or amniotic fluid samples collected in the same patient on the same day, were compared (Table II), there was a highly significant positive correlation between fasting plasma glucose levels and amniotic fluid glucos,e, p < 0.001 (Fig. l), and between amniotic fluid glucose andPOH butyrate values, p < 0.00 1 (Fig. 2). There was also a positive correlation between paired fasting plasma glucose and amniotic fluid /3-hydroxybutyrate levels, p C= 0.02 (Fig. 3). There was no significant correlation noted between paired maternal plasma glucose concentrations and amniotic fluid lactate or glycerol values nor between the levels of individual amniotic fluid solutes other than the P-hydroxybutyrate-glucose correlation already mentioned. These data indicate that increases in plasma glucose were accompanied by corresponding increases in amniotic Huid glucose and /3-hydroxybutyrate. Table III compares maternal fasting plasma glucose (FPG) levels with mean levels of the amniotic fluid solutes. Statistically significant increases in mean amniotic fluid glucose values occur in samples collected from borderline metabolic control (FPG 100 to 149 mg/lOO ml) and poor metabolic control (FPG 2 150 mg/lOO ml) groups. The mean /3OH butyrate concentration is fourfold increased in women with fasting plasma
Lactate
Wq&
POH butyrate
WWL)
11.0
0.21
9.6
0.13
Glycerol (mgllO0 ml) 201.2 -
sugars 2 150 mgi 100 ml, however, the limited number of observations and large standard deviations resulted in P values above the significant range. There was no significant change in the mean amniotic Huid glycerol or lactate levels in any of the categories grouped by maternal fasting plasma glucose levels, although slight increases in mean amniotic Huid lactate and glycerol concentrations are noted with higher maternal serum fasting glucose levels. Neonatal respiratory distress was associated with slightly higher tnean maternal fasting plasma glucose and amniotic levels of the above solutes, but only an approximately fourfold increase in mean /3OH butyrate approached significance (p = 0.09) by the nonparametric Mann-Whitney test (Table IV). The development of hypocalcemia, or hypoglycemia in the neonate correlated poorly with maternal fasting plasma sugar or amniotic Huid levels of glucose, lactate, glycerol, or P-hydroxybutyrate. Two perinatal deaths occurred in this series, both in the newborn period (Table V). In these two cases, except for small glucose increases and an elevated glycerol value in one instance, amniotic Huid solute levels were not markedly abnormal.
Comment It has been shown that amniotic fluid is principally a product of exchanges with the fetal circulation in the
892
Seeds et al.
last trimester of gestation and that lvater-soluble substances crossing from mother to fetus in significant quantities will appear in the fetal urine within several hours and thus enter amniotic Huid.” Therefore, increased maternal plasma glucose, ketoacid, and glycerol concentrations in poorly controlled pregnant diabetic patients will lead to elevated amniotic fluid concentrations of these compounds if they cross the placenta in significant quantities. The present preliminary study presents statistically significant data (Table II) indicating that maternal hyperglycemia (presumably on occasion associated M.ith ketoacidosis) can be accompanied bp increased ketoacids as well as glucose in amniotic fluid. Such findings correspond to previous reports of placental glucose transfer and indicate placental POH butyrate transfer since the hyperglycemic, hyperinsulinemic fetus of the diabetic mother would be a poor primary source for ketogenesis. This work then furnishes evidence for the first time of ketoacid transfer across human placental tissue in vivo in diabetes. However, clearly the positive correlation between elevated maternal fasting plasma sugar and amniotic glucose is much stronger (r =0.624) than that betM,een increases in maternal fasting plasma sugar and amniotic Huid j3OH butyrate (r = 0.294. Table II, Figs. I and 3). In fact, a rank correlation between these latter variables is insignificant (rs = 0.04, p > 0.05) reHecting the dependency of this statistically significant correlation on several high solute values. These findings undoubtedly reHect the absence of. significant maternal serum ketonemia except at high fasting plasma sugar levels. Thus, in this series of pregnant diabetic women. under medical supervision in a university center, marked maternal hyperglycemia accompanied by ketoac,idosis and elevated plasma glycerol levels was uncommon, thereby rc-ducing the chance of increased amounts of these substrates getting into amniotic fluid. The positive correlation between amniotic fluid ~ILIcase and P-h~droxyhuty’ate concentrations in paired samples (r = 0.545. Table II and Fig. 2) in this stud) further suggests that these solutes cross to the fetus and penetrate the amniotic cavity somewhat in parallel. Previous work from this laboratory, pert’ormecl with human placental tissue in vitro, indicated significant permeability of’ this tissue to glucose. P-hydroxybutyrate, and glycerol conforming to the relative molecular size and charge of these water soluble solutes.“” I‘hus, the present study, together with pt-evious in \ it ro arc consistent with the concept that investigations, human placental tissue is permeable not only to glucose but also to ketoacids. Poor metabolic control of diabetic
pregnancies tvitli maternal I-I)-perglycemii~ 1, 1bus Cl{ companied h\ increases iri amniotit fluitl glucose ant1 ketoacids. No significant increases in amniotic fluid lac-rate ot glycerol cotict.titrations occurred with matcrlial II\ perglycemia in this stud\. This obscwation i\ 01‘intct est particularly in view of the significant. iticrcase in amniotic Huid ketoacids in this situation. Iiowevct~, these findings do not preclude transfer of these solutes. Other possible explanations foi these data ~~oultl include: (1) No significant maternal ot- fetal lactate accumulation occurred. since there may have bee11 no significant maternal or fetal hypoxia accompanying the maternal li~~pergl~cemia and ketoacidosis. (2) ‘I‘here may have been no large maternal plasma glvccrol elrvation iii these patients. Absence of amniotic fluid glycerol increases are diffjrult to e\Taluate in the absence of tnaternal plasma gl!,ccrol measurcmel~ts. (3) Glycerol ma! cross the placenta, enter and leave the amniotic cavity in difftrent chronologic sequence than the other solutes, and thus increases in amtliotic fluid glycerol concentrations occurred in these patients but were not present at the time of amniotic fluicl collections. (4) The hyperinsrtlinemic fetus; of t hc diabetic mother may utilize glycerol for lipogenesis and thus not contribute excess amounts of this compound to amniotic Huid even during a period ot increased glycerol transfer from the mother. Finally. This study demotistl-dted lirnited clinical usefulness for these amniotic Huid solute determinations in this gro~rp of well-controllecl diabetic patienb. Small increases in maternal plasma glucose. amliiotic Ruid glucose, glycerol. and lactate and a larger increase irl amniotic fluid /K)H buryrate occurred in pregnancies resulting in neonatal I-espiratcq distress syndrome (Table IV) but were not statistically significant-. ‘l‘he lack of cot-relation of amniotic fluid solute levels to fetal h~lwglpcw~~ia or hypocalcemia most likel! also rcflectrd intensive management of these high-risk tteonates where immediate electrolyte and glucose parenter-al alimct~tatioti \vas used to reduce the incidence of’ these neonatal ha~ai-ds. 7‘his poor correlation 7vas not unexpcc ted in view of the relatively good metabolic control furnished these pregnant women and the small number of neonatal c-omplications. It is difficult to demonstrate a relationship between elevated levels of’ these amniotic fluid solutes ancl neonatal complications when there is a relativeI! small incidence of each oc‘l‘he authors are indebted to Dale Bucalo and Bernadette Plair for their t.echnical assistance and to Jane Holvroyde for statistical consultation.
Volume
135
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Changes in amniotic glucose, pOH butyrate, glycerol, and lactate 893
REFERENCES
1. Persson, B., ;i.nd Lunell, N. 0.: Metabolic control of diabetic pregnancy. Variations in plasma concentrations of glucose, free fatty acids, glycerol, ketone bodies, insulin, and human chorionic somatomammotropin during the last trimester, AM. J. OBSTE:T. GYNECOL. k!2:737, 1975. 2. Oaklev. , N. W.. Beard. R. W.. and Turner. R. C.: Effect of sustained maternal hyperglycemia on the fetus in normal and diabetic Ipregnancies, Br. Med. J. 1:466, 1972. 3. W iddas, W. F.: Transport mechanisms in the fetus. Br. Med. Bull. 17*107 1961. ., 4. James, E. J., Raye, J. R., Gresham, E., Makowski, E. L., Meschia, G., and Battaglia, F. D.: Fetal oxygen consumption, carbon dioxide production, and glucose uptake in a chronic sheep preparation, Pediatrics 50:361, 1972. 5. Drazancic, A., and Kuvacic, I.: Amniotic fluid glucose concentration, AM. J. OBSTET. GYNECOL. 120:40, 1974. 6. Wood, G. P.. ;and Sherline, I). M.: Amniotic fluid glucose: A maternal, fetal, and neonatal correlation, AM. J. OBSTET~ GYNECOL. 122:151, 1975. 7. Archimant, G., Gelizan, J. M., Ross, R. A., and Olthshe, 0.: Glucose concentration in amniotic fluid: Its possible significance in diabetic pregnancy, AM. J. OBSTET. GYNECOL. ll!k596, 1974. 8. Cassady, G., Blake, M., Bailey, P.. Younger, B., and Summers, J. Amniotic fluid glucose in pregnancies GYNECOL. complicated with diabetes, AM. J. OBSTET. 127:21, 1977. 9. Spellacy, W. N., Buhi, W. C., Bradley, B., and Holsinger, K. K.: Maternal, fetal and amniotic fluid levels of glucose, insulin and growth hormone, Obstet. Gynecol. 41:323, 1973. 10. Seeds, A. E.: Water dynamics in the amniotic Huid in Fair-weather, D. V. I., and Eskes, T, K. A. B., editors:
Discussion C. COODLIN, Omaha, Nebraska. Dr. Seeds is a recognized authority on the formation of amniotic fluid, and I have enjoyed and profited from reading his interesting paper. However, there are some features of the paper which I do not understand. Maternal ketones are considered to pass the placenta and to be a possible source of fuel for the fetus.’ I am puzzled by the authors’ statements that they “furnished evidence for the first time of ketoacids transfer across human placental tissue in vivo in diabetes.” Did the authors suspect that placental transfer would be different in diabetes or have they demonstrated a new observation that I missed? Dr. Seeds and colleagues believe that amniotic fluid solute concentrations reflect exchange with fetal circulation. I do no-t believe that anyone has proved this relationship to the exclusion of fetal membrane transfer. It is these membranes which presumably maintain the amniotic Huid composition of many constituents in cases of fetal d,eath. Several years ago, I thought we demonstrated that the human amnion may inHuence amniotic Huid Ielectrolyte composition. Human fetal membranes will function like a toad bladder by responding to oxytocin and by alterating sodium and chloride gradients.* I reasoned that the amniotic Huid DR.
ROBERT
Amniotic Huid-Research and Clinical Application, Amsterdam, 1972, Excerpta Medica. 11. Battaglia, F. C., and Meschia, G.: Principal substrates of fetal metabolism, Phvsiol. Rev. 58:499, 1978. 12. Kim, Y. J., and Felig, P.: Maternal and amniotic Huid substrate levels during caloric deprivation in human pregnancy, Metabolism. 21:507, 1972. 13. Paterson, P., Sheath, J., Taf‘t, P., and Wood, C.: Maternal and fetal ketone concentrations in plasma and urine, Lancet 1:X62, 1967. 14. Sabata, V., Wolf, H., and Lausmann, S.: The role of free fatty acids, glycerol, ketone bodies, and glucose in the energy metabolism of the mother and fetus during delivery, Btol. Neonate 13:7, 1968. 15. Smith. A. L., and Scar&n, J.; Amniotic fluid D (-) @-hydroxybutyrate and dysmature newborn infant, AM. 115:569, 1973. J. OBSTET. GYNECOL. 16. Felig, P., and Lynch, V.: Starvation in human pregnancy: Hypoglycemia, hypoinsulinemia, and hyperketonemia, Science 170:990, 1970. 17. Hoehorst, H. J.: L(+)-Lactate: Determination with lactic dehydrogenase and DPN, in Bergmeyer, editor: Methods in Enzymatic Analysis, New York, 1963, Academic Press, Inc., pp. 266-270. 18. W illiamson, D. H., Mellanhy, J., and Krebs, H. A.: Enzymatic determination of D (-) Beta-hydroxybutyric acid and aceto acetic acid in blood, Biochem. J. 82:90, 1962. 19 Pinter, J. K., Hayashi, J. A., and Watson, J. A.: Enzymic assay of glycerol, dihydroxyacetone and glyceroldehyde, Arch. Biochem. Biophys. 121:404, 1967. 20. Seeds, A. E., Leung, L., Clark, K., Stys, S., and Russel, P. T.: Permeability of chorion laeve in vitro to POH butyrate and glycerol. Suhmitted for publication.
of the anencephalic fetus might have electrolyte composition different from that of the normal fetus, since these fetuses often lack a pituitary gland. I have looked in vain for these changes in amniotic Huid electrolyte concentrations, but Pettit, King, and Blau” found the amniotic Huid of anencephalic fetuses to be very low in glucose. W e subsequently have analyzed the amniotic Huid of two anencephalic fetuses that were at 34 to 36 weeks’ gestation without severe hydraxnnios. In one, the fetus was alive at birth. In both cases, the amniotic Huid glucose was so low as not to be measurable, and in the live fetus its cord blood sugar was 36 mgi100 ml. Since fetal urine and tracheal Huid contain little or no glucose, I assume that fetal membranes must be a significant source of the amniotic Huid glucose. Amniotic Huid glucose concentration is known to decline in value with gestation, and to be very low in cases of chronic fetal distress and postmaturity. This decline perhaps reflects the increase in fetal swallowing and digestion described in these conditions,4 and its relative absence in the anencephalic fetus might indeed represent fetal hormonal effects on the amnion. Given the few exceptions of fetal disease as listed above, 1 think that we can assume that amniotic Huid glucose values, when corrected for gestation, reflect maternal blood sugar values. Since the authors’ paper gives LIS maternal data
894 Seeds et al.
onl! about fasting blood sugars, it is not possible lo conti1111e the argn1nent over whether the in viva fetus (or its membranes) is controlling the amniotic f-l&l levels of’ those solutes measured by the authors. Perhaps Dr. Seeds has more data on these solutes relativc to aninion transfer or maternal/fetal values. hly third question concerns Dr. Seeds’ assumption that 1he l’ctuses in this study all had increased insulin levels. (Znrrent dogma considers the fetus of the diabetic mother to have hyperinsulinemia,” but would this be true in those whose mothers are under the “good metabolic control” claimed 1~) the authors? ,Anlniocentesis is a commonly done procedure in high-risk clinics. I know that 1 have ordered all available laboratory tests on amniotic Huid in high-risk cases in hopes of finding an abnormalit\. that could have signilicance as far as fetal welfare is concerned. Howe\w, in the absence of si1nnltaneous fetal-maternal blooct studies, I think this sort 01’activity is unlikely to product significant acl~aricenicnt in fetology. There are exceptions, but most of out- useful laboratory testx of amniotic. ttuid constitnents were based on a prio1 deniotist~ation of their significant elevations in the concerned fetus. In m) opinion, Dr. Seeds a11d associates paper \\ould have more significance if siniultaneous fetal and maternal blood data were available f’or the solutes the\. 111easuld REFERENCES 1. 2.
3.
F&g, P.: Body fuel metabolism and diabetes in pregnancy. Med. Clin. North Am. 61:43. 1977. Goodlin, R. C., and Kresch, A. J.: Amniotic fluid osmolality following intra-amniotic injection of 23% saline, AM. J. OBSTET.GYNECOL.100:839, 1968. Yrttit, B. R.. King, G. S., and Blau, K.: Low glucose concentration in amniotic Huids from anencephalic pregnancy, Lancet 2: 1288, 1977.
‘1.
hlclain, C. R.: Amniography studies of the gastrointestinal motility of the human fetus, AM. J. OBSTET GYNECOL. 86: 1079. 1063.
I5
Adam, P. A.: Infant of a diabetic mother: Energy imbalance between adipose tissue and liver, Sem. Perinatol. 2.929 a. ., 197x.
DR. ALLAN B. WEINGOL~~, Washington, D. C. Dr. Seeds and his colleagues have addressed a most interesting and important subject in attempting to correlate amniotic Huid ketone levels with maternal glucose COIIcentration as a reflection of diabetic metabolic control. Current clinical practice calls for validation of fetal maturity prior to termination of a diabetic pregnancy. thereby providing us with one or ttiore sampies of arnniotic fluid prior to delivery. Utilization of this fetal solution f’w- predelivtiry alteration in therapy or for prediction of perinatal complications is a valid objective. The paper also serves to enhance our understanding of‘ the kinetics of ketone transfer across the placenta and draws the appropriate conclusions that meternal ketones are passed to the fetus and thus gain access to
tlie ami1iotic tluid. ‘I‘hc tlitIic.al illlpOlTilll~~C 01 Lchtosis Itt diabetic pregnant\‘. tiowt’wr, c~\-rcwds I,r\ontl tlw immediate association wirh feral cll~i1tl1i11 u1crc) 01. IICOnatal death. In 1069 Church,ll, Be~wtth al~tl Scnlorc.’ rcpxIing on the tle~elopment of infants of diabetic- mothers (1DN) from the I’erinatal (Mlaboratiw PIY+Y~I, 1101etl that the presenw ofa~~to1iu1-ia during pregnancy ws ;I critical variable itifluenciiig the intcltrctu.1l status of IDM’s at 4 years ot age. Inteltcctual inip3irnifnt \\as more often present in offspring whosc~ tiiotliei~s had displayed ;~cctoiinria, On the orticr I~i~tid. inlcllccti1al stat115 was unrelatecl to [Vhite’s classiticatioll, gcstational age, or the amount ot insulin rcwi\ctl I)\ the mother during prrgnanc! In 195’i. Stchbcns. Bakw. ;111d Kitchill” c onfirtnetl these results in a prosptt.tiw sruti\ on an rnlirelv different popiilatioti. .l‘tica (Iif’trcnce bct\vwti IQ scow at .5 years of age 01’children born to aceto11c-iieaati\e is. ;icetone-l,ositi\.e morhcrs \+.assignificanr at p < 0.05. It is important to nolc that 1he caust’ 01‘awtot1~lria \\‘a> not specified in eittrcr stIlti>. It ina? haw rc~prcsentcd true diabctit kctoacidosis scw~ntlar~ to poor control (insiifficicnt insulin or cxc es5 calories) or thr pregnatic\ rnhariced tcntlrnc-\ to starvation ketosis sccontl;~r~ to insufficient calories. I’hc critical itnportaticc ot adcquate niaternat caloric inrake in diahetit pregnanc\~ with entphasis 011 coniples ~a1boh~dratcs ~arrnot be ovcret11l)liasi~~~l.” For exi1nlple, nlater11al bt~~~~tllevels ot beta-l1~dr~~x~buqrate and acetoacetic. acid are tl\o 10 f’our times greater in pregnant>’ al‘tc.1.o\crnight fasting than in the nonpregnant state.’ Maternal ketonemia is associated with ketone body acc11mulation in the amniotic flnid and, bp inference, s11ggesfs that the fc*tus is exposed to substances that tuav ha\~ an atl~erae et’l’ec~~on subsequent 11twr&eha\ ioral de\ clopment. I‘ptake trf ketone ac.ids has her11 observed in the human fetus and the enzymes f’or ketone oxidation have bee11 idcntilied in fetal brain tissne.” Perhaps 1his is tl1e major problem with Dr. Seed’s syndy. it ma) be a USC OF wrong time at1d wrong place. Talented physicians. managing overt diabetes in pregnancy, who generally achieve their objective of eugl~cemia, are simpty not going to see significant l~laternal ketoacidosis. Nevcrt helcss, how much mow inform+ tive this paper ww1ld ha\ e hecn if’ maternal ketones had been studied and paired \\+th amniotic fiuid lwels. A comparison of maternal filu~oseihydrox~b~~t~r~1t~ levels VS. amniotic levels of the same componnds ~onld have frilly excluded a starwtion ketosis cwnptment and may Ivet have clarilicd rhe var>’ large standard dcviation disptaycd in the beta-hvtl1-o?cybutyrate levels. TM) first question, then, of Dr. Seeds is “Cl’as there a specific reason ~rhy maternal levels of1he solutes stndied, other than glucose, were not rneasurecl?” A second cIuestio1i relates to another substance which Inay reflect glucose metabolism a11d has brcrl (‘1 altla1Nl
Volume 135 Number 7
Changes in amniotic glucose, p0l-i butyrate, glycerol, and lactate 895
by amniotic fluid analysis, namely, insulin. Amniotic fluid insulin appears to be totally fetal in origin as evidenced by the failure to measure 112”labeled insulin in that compartment after maternal infusion. Levels of greate;. than 60 FLU/ml, particularly in association with low amniotic Iluid levels ol‘ glucose, correlate wel! with poor perinatal outcome. ’ Dr. Seeds, could your studies be extended to include insulin assessment? Do you believe that amniotic ketones are a IIKX~ accurate “memory” statement about pre-existing metabolic control than insulin? REFERENCES
Churchill, J. A., Berendes, H. W.. and Nemore, J.: AM. J. ~BS1.E’~. GYPIECOL. 105:257, 1979. Stehbens, J. A., Baker, G. L., and Kitchell, N.: AM. J. ORSTET. GYNECOL. 127:408, 1977. Weingold, A. B.: Diabetes in pregnancy, pg. 163 in Caplan, R. M., and Sweeney, W. J., editors: Advances in.Obstet and Gynecol, Baltimore, 1978. The Williams & Wilkins Company. 4. Felig, P., and Lynch, V.: Science 170:990, 1970. 5. Page. M. A.. and Williamson, D. H.: Lancet 2:66, 197 1. 6. Newman, R., and Tutera, G.: Obstet. Gynecol. 47:599, 1976. DR. SEEDS (Closing). Dr. Goodlin’s series of questions reflect his understanding and expertise in this field, and his innovative thinking about this area. His statement about the lack of significance I would like to modify to make clear that although limited clinical usefulness of this study was demonstrated, there was a clear statistically significant relationship demonstrated between increases in amniotic Huid, betahyclroxybut~ratc, and increases in maternal serum glucose. T-his strong relationship provided firm evidence in the human subject that beta-hydroxybutyrate is crossing the placenta from the mother since the fetus would not be producing this compound in excessive amounts in the presence of high insulin levels. Moving on to some of the other points that Dr. Goodlin brought up, and they are all interrelated, my under-standing of amniotic fluid formation is pure and simple. It is largely a product of exchanges with the fetus and incleascs in solutes in amniotic fluid, with very few exceptions, very likely parallel increases of the same solute, in the fetus. If solute concentrations do not increase in the fetus, they do not get into the amniotic fluid. For this reason I think this kind of evidence, i.e., amniotic fluid increase in beta-hydroxybutyrate, as well as glucose, reHects maternal transfer of these solutes across the placenta to the fetus. I was as surprised as Dr. Goodlin, having read a number of textbook statements implying that, of course, ketoacids cross the placenta, and accepting this as fact and mechanism, for the great threat to th,e fetus represented by maternal acidosis in diabetes. However, extensive review of the literature failed to provide evidence supporting this concept. Theoretical reasoning
suggests that ketoacids might not cross the placenta since they have a low pK (3 to 4) and are largely ionized at physiologic pH. Because of such physical chemical characteristics, these compounds could be expected to cross placental tissue with difficulty. Now, looking at evidence in the literature for the transfer of these solutes from mother to fetus, in the sheep they do not cross at all. In the human subject it seems very clear to me that all available data indicating transfer were obtained where the mother was fasted and the fetus then hypoglycemic and hypoinsulinemic. Tht fetus could thus be producing these ketoacids and not accumulating them by placental transfer from the mother. Under these conditions, small parallel rises in maternal and fetal ketoacids do not provide strong evidence of placental transfer. I looked for data on cord blood or amniotic fluid increases in ketoacids during the diabetic pregnancy, where the hyperglycemic hyperinsulinemic fetus would not be a primary source of ketoacids, and there is limited or zero evidence in that area, and hence this study. It is my contention that this study then for the first time provides evidence in pregnant diabetic subjects of maternal to fetal ketoacid transfer. Clearly this study would be stronger if simultaneous maternal serum ketoacids had been measured, as both Drs. Goodlin and Weingold suggested. One reason why this was not done is that about half of this study was retrospective. ive had the fluid collected in these cases before planning these analyses. A prospective study is now under way in which maternal serum and amniotic fluid ketoacid glycerol and free fatty acids and amniotic fluid insulin, glucsgon, and C-peptide are being measured. We will try to answer these kinds of questions. Dr. Goodlin mentioned fetal membrane function as possibly similar to toad bladder, and cited experiments with oxytocin, where in vitro permeability of placental membranes was apparently changed. Such changes might influence amniotic fluid values and we cannot exclude that possibility. However, in our laboratory, 5 or 6 years ago, we did study the effect of oxytocin vasopressin and 3’5’AMP on placental membranes to see if there was an effect similar to that found with toad bladder where pore size and membrane permeability are affected and there is increased permeability to solutes and water. In a large over a \vide concentration number of experiments, range of these agents, we found no in vitro response or change of in vitro placental permeability in human subjects. Finally, regarding Dr. Weingold’s excellent remarks, I agree that amniotic Huid insulin measurements yould improve the study. Amniotic fuid insulin is probably of fetal origin and may well provide a better memory system then ketoacids. For this reason our prospective study includes measurements of insulin, C-peptide, and glucagon.
A.
ELMORE
LIM M.
!i.
SEEDS,
LEUNG,
WILSON
PAUL, Cincinnati.
T.
M.D.
M.S.
TABOR, RUSSELL,
PH.D. PH.D.
Ohio
Amniotic fluid glucose. POH butyrate, glycerol, and lactate concentrations were measured in 75 samples collected in the third trimester of pregnancy from 50 diabetic patients, all but four of whom required insulin. Increases in maternal fasting plasma sugar were accompanied by corresponding increases in amniotic fluid glucose and on.occasion increases in amniotic fluid pOH butyrate. These data correspond to previous reports of placental glucose transfer and in addition, provide statistically significant evidence of placental @OH butyrate transfer since the hyperglycemic, hyperinsulinemic fetus of a diabetic mother would be a poor primary source for ketogenesis. Relatively poor correlation of elevated fluid levels of these solutes to fetal outcome probably reflects a low incidence of maternal hyperglycemia, ketoacidosis, and over-all reduced neonatal morbidity-mortality rates in this group of metabolically well-controlled, predominantly insulin-requiring diabetic patients managed in a regional high-risk perinataf center. (AM. J. OBSTET. GYNECOL. 135:887, 1979.)
PROPER METABOLIC control of diabetic pregnancy results in maternal blood sugar in the euglycemic range,’ thus preventing increases in plasma ketoacids and glycerol that accompany hyperglycemia in this diseased state. Clinical evidence for proper levels of these solutes is usually obtained by periodic blood sugar measurements and/or determination of urine suger and ketone bodies. It is common in the routine management of the insulin-requiring diabetic pregnancy to perform an amniocentesis late in the third trimester for purposes of assessing fetal maturity. It was felt that perhaps determination of amniotic fluid levels of glucose, glycerol, ketoacids, and lactate could also be helpful in the clinical assessment of the diabetic pregnancy. For example, if elevation of these solutes in maternal
From the Department oJ Obstetrics and Gynecology, Uniuwsity of Cincinnati School of Medicine. Supported in part by United States Public Health Service Grant No. IPOL-HD-11725-01 and by a grant from the Juvrnile Diabetes Foundation. Presented by invitation at the One Hundred and Second Annual Meeting oj the American Gynecological Society, Hot Springs, Virginia, May 16-19, 1979. Reprint request.7:Dr. A. Elmore Seeds, Department d Obstetk-s and Gynrcology, University oJ Cincinnati School of Medzrine, Cincinnat~~, Ohio 45267. 0002-9378/79/230887+09$00.90/O
@I 1979 The C. V. Mosby Co.
circulation is an indication of poor metabolic control or improper management of the diabetic pregnancy and if these solutes cross the placenta in significant quantities to the fetus, it is reasonable to expect that amniotic fluid levels of these compounds might provide an indication of maternal metabolic status in this condition. Except for glucose, there are almost no data on amniotic fluid accumulation of these metabolically significant compounds in diabetic pregnancy. If these solutes cross to the fetus and get into amniotic fluid in significant quantities then depending on their relative ability to escape (i.e., turnover time or disappearance time) they may reflect events in maternal and/or fetal compartments for varying periods of time or function as a memory system. Previous studies have indicated that human placental tissue is readily permeable to glucose and that fetal serum levels parallel maternal levels over a wide concentration range, probably reflecting the facilitated diffusion of this solute.2-J Elevated amniotic fluid glucose concentrations have also been measured in diabetic pregnancies5-” in parallel with maternal hyperglycemia, indicating significant transport of this compound from mother to fetus to amniotic fluid. Comparisons between amniotic Huid glucose levels and perinatal and neonatal outcome have also been re887
888
Seeds et al
Table I. Maternal
fasting plasma glucose and atnniotic SOlUlf.~
Huid solutes for diabetic and nondiabetic
lhhtir
Plasma fasting glurose (mgi 100 ml) Amniotic fluid glucose (mgi100 ml) Lactate (mEq/L) ,kXBH butyrate (mEq/L) (ilyrerol (mgi 100 ml)
119.3 29.6 9.3 0.22 151.9
!Vontliab&
-t 60.8 (60) t 28.5 (7.5) t 3.0 (56) ‘-+ 0.38 (61) f 39.4 (45)
Not 12.5 8.6 0.19 134.4
mea&t,-ed r 6.4 (40) k 2.3 (33) ? 0.23 (25) 2 28.4 (IS)
pregnanc-jes* I’ r’alri? co.0 I ~0.05
-cO,1),5 >o.o:i
*Values given are mrans ? standard deviations. Numbers or observations are in parentheses. Table II. Correlation between paired plastna glucose and amniotic fluid solutes in diabetics* .4mnioticjluid C&mm
Fasting plasma glucose
Amniotic Huid glucose
Lactate @OH hutyrate
0.62!:
WN
Lactatr 0.08 (48) 0.02 (56)
WH
butymte 0.29t (53) 0.55$
(61)
0.04 (55)
Glywrol 0.10 (41) -0.01 (45) 0.03 (41)
-0.08
(44)
*Values given are correlation coefficients, Numbers of observations are compared in parentheses. tCorrelation significant at P < 0.02. fCorrelation significant at P < 0.00 1.
ported and, although interpretations differ, abnormally high amniotic fluid glucose levels appcarecl to correlate with increased perinatal morbidity and mortality rates. These previous studies are preliminary in that they have been performed on small numbers of‘ insulin-requiring diabetic pregnancies, although the incidence of‘ hyperglycemia was surprisingly high. apparently due to relatively inadequatr metabolic control. (:onflicting information from animal studies indicated that ketoacids crossed the placenta in rodents but did not penetrate the ungulate placenta.” There is no clear evidence documenting human placental permeability to these compounds. Increases in fetal ketoacids in the fasting pregnant state have been interpreted as evidence of placental transfer of. these solutes in the Presence of’ very small positi\c human subject.“-‘” umbilical venoarterial differences in POH butyratc concentration and correlations bet\vcen increasing maternal and fetal blood Levels of‘ this metabolite were suggestive hut not conclusi\~e indications of placental transfer. However, when tnaternal and fetal compartments are hypoglycemic and hypoinsulinemic~ and pt-rsumably. then, both ketogenic sources, such increases do not necessarily indiratr placental transfer of ketoacids. No tneasurements have been recorded in the diabetic pregnancy wherein the mother is hyperglycemic arid hypoinsulincmic and the f’etus hyperglycemic with
adequate or increased amounts of iltsulin (thus increased fetal ketoacids ~vo~11d presumahly he of‘ matet.nal origin) to show that elevated ketoacids itt the maternal compartment arc transtnitted across,the placenta to the fetus. Excess lactate will accumuIate in maternal and/or fetal c.otnpartmcnts in response to hypoxia and the possibility exists that increased fetal lactate ma) lead to increased amniotic fluid levels of‘this solute during acute or chronic distress accompanying poor maternal metabolic control of diabetes. Evidence f’w transplacental passage of’ glycerol in animal or man is inconclusive. Thus with the exception of glilpose, little is known about the placental transfer and amniotic fluid accumulation of these solutes which are possibly elevated in the pregnant diabetic \+oman. Therefhrc-, measurements 01’amniotic fluid glucose, P-hvdrt,xybrltyt-air. glycerol, and lactate in diabetic pregnancies were undertaken to determine if these levels wwe altered in thr diabetic pregnancy and, if’ so, did these changes tol-relarr to maternal blood sugar levels and/or fetal ottwmw.
ftllelhodology Sixty amniotic fluid samples were obtained in the late third trimester from 38 pregnant diabetic patients. Maternal fasting blood sugar was measured on the morn ing of each amniocentesis. An additional 15 amniotic fluid satnples were obtained in 12 diabetic patients wherein a maternal fasting blood sugar was not collected. These 50 pregnant diabetic pzatients were distributed according to the White classification as fi)llwvs: Class A, 4; Class B, 14: Class C, 2.5; Class I). 4; Class K, 3. Where sufficient amniotic fluid volume was available, amniotic fluid glucose, P-hydroxybutyratc, glycerol, and lactate concentrations were determined and compared with values collected from 40 nondiabetic control pregnancies at the same gestational age. The p-hydroxybutyriciacetoacetic acid ratio is usually maintained or increased during the rise in ketone bodies accompanying poorly controlled diabetes.‘” Thus dctermination of P-hydroxybutyrate as a valid indicator of ketoacid accumulation in this condition seemed appropriate. Glucose \vas measured en7vmatically l)\
Volume
135
Number
7
Changes in amniotic glucose, pOH butyrate, glycerol, and lactate
889
250 -. 8 s $j 0 3
225
.
200 175 150 -
3 125 8 a 100 G” b8 -75 --
.
s
.
T
50 :25 0 0
. :
. -.
. . .z ’ . . .2.
l
. . .;*&. ;; $2 .* * 1 I I I I I I ; 50 loo 150 200 250 300 350 400 FASTING PLASMA GLUCOSE (mg%)
I 450
I 500
CORRELATION Id= 0.62 PcO.001 RANK CORRELATION (r,l=0.70 PCO.001
Fig. 1. Amniotic fluid glucose concentrations are plotted vs. maternal fasting plasma glucose concentrations. The correlation and rank correlation are given for this relationship.
25
50
I I 175 75 100 125 150 AMNIOTIC FLUID GLUCOSE(mg%)
I 200
I 225
I 250
CORRELATION (r)=.55 PO.O5
Fig. 2. Amniotic fluid POH butyrate concentrations are plotted vs amniotic fluid glucose concentrations. The correlation and rank correlation are given for this relationship. means of a glucose oxidase method. Amniotic fluid lactate was measured enzymatically according to the method of Hoehorst.” P-Hydroxybutyrate was measured according to the method of Williamson, Mellanhy, and Krebs. I8 Glycerol was measured as described by Pinter, Hayashi, and Watson.‘g All patients were hospitalized at the University of Cincinnati Medical Center Hospitals for predelivery care and metabolic control at the time of amniocentesis. Amniocenteses were performed from 0 to ‘Ll days prior to delivery. Most of these patients, benefiting from contemporary metabolic management of the diabetic pregnancy, were in reasonably euglycemic range.
Amniotic fluid solute values were correlated with fetal outcome in an attempt to evaluate the prognostic significance of these measurements in the diabetic pregnancy. Neonatal morbidity was defined by the following criteria: Respiratory distress syndrome (RDS)-grunting, tachypnea >60 minute retractions, cyanosis w-ithout added oxygen: all of these signs present for more than 6 hours Respiratory distress-tachypnea: >60/min Hypoglycemia-serum glucose: 530 mgi 100 ml Hypocalcemia-serum calcium: 57 mgi 100 ml for premature infants 58 mg/ 100 ml for term infants
890 Seeds et al.
\
3.0
2 lu g
27 241
it
18-
3&
15-
bF 21c
Q a 3
09-
12-
’ 2 Q
060.3-
$
0.0 0
.
.
** . .
-4 . 22X? :; 1: . : 1.:..I -** 1 . I. I I I loo 150 200 250 300 350 400 50 FASTlNG PLASMA GLUCOSE hq%)
I 450
1 500
CORRELATION (r)=.ZS Ptlae WdL)
Cl00 2100
18.8 2 9.4 (32) 45.0 -1-39.9 (28) p < 0.01
153.2 -1-39.5 (22) 153.8 2 42.5 (19) N.S.
0.15 2 O.OS(25) 0.28 k 0.59 (28) N.S.
9.1 * 2.1 (25) 9.6 AT4.1(23) N.S.
Cl00 100 S 0.05
P-o.05
24.5 k 14.5 (38) 32.5 k 18.5 (7)
>0.05
>0.05
Yes No Yes
9.6 t 4.0 (25) 9.9 T 2.5 (7)
BO.05
>0.05
No
0.13 ? 0.06 (27) 0.51 _’ 0.93 (7)
>0.05
20.05
z-o.05
P-o.05
NO
Amniotic Huid lactate (mEq/L)
fluid solute concentrations
Yes No Yes
143.1 k 48.9(18) 154.7 2 27.8 (6)
891
*Values given are means f standard deviations. Numbers of incidence are in parentheses. Table V. Amniotic
fluid sollute and maternal
Cause of &ath 1. RDS 2. Respiratory disease
fasting plasma glucose values in two cases Amniotic fluid
Maternal ,fa.sting plasma glucose
No. of days separating amniocentesis from deliveq
Glucose (mgllO0 ml)
97
3 20
17.6 25.3
tate, /3-hydroxybutyrate, and glycerol concentrations in the diabetic compared to the nondiabetic group were not statistically significant. However, when paired samples, i.e., plasma or amniotic fluid samples collected in the same patient on the same day, were compared (Table II), there was a highly significant positive correlation between fasting plasma glucose levels and amniotic fluid glucos,e, p < 0.001 (Fig. l), and between amniotic fluid glucose andPOH butyrate values, p < 0.00 1 (Fig. 2). There was also a positive correlation between paired fasting plasma glucose and amniotic fluid /3-hydroxybutyrate levels, p C= 0.02 (Fig. 3). There was no significant correlation noted between paired maternal plasma glucose concentrations and amniotic fluid lactate or glycerol values nor between the levels of individual amniotic fluid solutes other than the P-hydroxybutyrate-glucose correlation already mentioned. These data indicate that increases in plasma glucose were accompanied by corresponding increases in amniotic Huid glucose and /3-hydroxybutyrate. Table III compares maternal fasting plasma glucose (FPG) levels with mean levels of the amniotic fluid solutes. Statistically significant increases in mean amniotic fluid glucose values occur in samples collected from borderline metabolic control (FPG 100 to 149 mg/lOO ml) and poor metabolic control (FPG 2 150 mg/lOO ml) groups. The mean /3OH butyrate concentration is fourfold increased in women with fasting plasma
Lactate
Wq&
POH butyrate
WWL)
11.0
0.21
9.6
0.13
Glycerol (mgllO0 ml) 201.2 -
sugars 2 150 mgi 100 ml, however, the limited number of observations and large standard deviations resulted in P values above the significant range. There was no significant change in the mean amniotic Huid glycerol or lactate levels in any of the categories grouped by maternal fasting plasma glucose levels, although slight increases in mean amniotic Huid lactate and glycerol concentrations are noted with higher maternal serum fasting glucose levels. Neonatal respiratory distress was associated with slightly higher tnean maternal fasting plasma glucose and amniotic levels of the above solutes, but only an approximately fourfold increase in mean /3OH butyrate approached significance (p = 0.09) by the nonparametric Mann-Whitney test (Table IV). The development of hypocalcemia, or hypoglycemia in the neonate correlated poorly with maternal fasting plasma sugar or amniotic Huid levels of glucose, lactate, glycerol, or P-hydroxybutyrate. Two perinatal deaths occurred in this series, both in the newborn period (Table V). In these two cases, except for small glucose increases and an elevated glycerol value in one instance, amniotic Huid solute levels were not markedly abnormal.
Comment It has been shown that amniotic fluid is principally a product of exchanges with the fetal circulation in the
892
Seeds et al.
last trimester of gestation and that lvater-soluble substances crossing from mother to fetus in significant quantities will appear in the fetal urine within several hours and thus enter amniotic Huid.” Therefore, increased maternal plasma glucose, ketoacid, and glycerol concentrations in poorly controlled pregnant diabetic patients will lead to elevated amniotic fluid concentrations of these compounds if they cross the placenta in significant quantities. The present preliminary study presents statistically significant data (Table II) indicating that maternal hyperglycemia (presumably on occasion associated M.ith ketoacidosis) can be accompanied bp increased ketoacids as well as glucose in amniotic fluid. Such findings correspond to previous reports of placental glucose transfer and indicate placental POH butyrate transfer since the hyperglycemic, hyperinsulinemic fetus of the diabetic mother would be a poor primary source for ketogenesis. This work then furnishes evidence for the first time of ketoacid transfer across human placental tissue in vivo in diabetes. However, clearly the positive correlation between elevated maternal fasting plasma sugar and amniotic glucose is much stronger (r =0.624) than that betM,een increases in maternal fasting plasma sugar and amniotic Huid j3OH butyrate (r = 0.294. Table II, Figs. I and 3). In fact, a rank correlation between these latter variables is insignificant (rs = 0.04, p > 0.05) reHecting the dependency of this statistically significant correlation on several high solute values. These findings undoubtedly reHect the absence of. significant maternal serum ketonemia except at high fasting plasma sugar levels. Thus, in this series of pregnant diabetic women. under medical supervision in a university center, marked maternal hyperglycemia accompanied by ketoac,idosis and elevated plasma glycerol levels was uncommon, thereby rc-ducing the chance of increased amounts of these substrates getting into amniotic fluid. The positive correlation between amniotic fluid ~ILIcase and P-h~droxyhuty’ate concentrations in paired samples (r = 0.545. Table II and Fig. 2) in this stud) further suggests that these solutes cross to the fetus and penetrate the amniotic cavity somewhat in parallel. Previous work from this laboratory, pert’ormecl with human placental tissue in vitro, indicated significant permeability of’ this tissue to glucose. P-hydroxybutyrate, and glycerol conforming to the relative molecular size and charge of these water soluble solutes.“” I‘hus, the present study, together with pt-evious in \ it ro arc consistent with the concept that investigations, human placental tissue is permeable not only to glucose but also to ketoacids. Poor metabolic control of diabetic
pregnancies tvitli maternal I-I)-perglycemii~ 1, 1bus Cl{ companied h\ increases iri amniotit fluitl glucose ant1 ketoacids. No significant increases in amniotic fluid lac-rate ot glycerol cotict.titrations occurred with matcrlial II\ perglycemia in this stud\. This obscwation i\ 01‘intct est particularly in view of the significant. iticrcase in amniotic Huid ketoacids in this situation. Iiowevct~, these findings do not preclude transfer of these solutes. Other possible explanations foi these data ~~oultl include: (1) No significant maternal ot- fetal lactate accumulation occurred. since there may have bee11 no significant maternal or fetal hypoxia accompanying the maternal li~~pergl~cemia and ketoacidosis. (2) ‘I‘here may have been no large maternal plasma glvccrol elrvation iii these patients. Absence of amniotic fluid glycerol increases are diffjrult to e\Taluate in the absence of tnaternal plasma gl!,ccrol measurcmel~ts. (3) Glycerol ma! cross the placenta, enter and leave the amniotic cavity in difftrent chronologic sequence than the other solutes, and thus increases in amtliotic fluid glycerol concentrations occurred in these patients but were not present at the time of amniotic fluicl collections. (4) The hyperinsrtlinemic fetus; of t hc diabetic mother may utilize glycerol for lipogenesis and thus not contribute excess amounts of this compound to amniotic Huid even during a period ot increased glycerol transfer from the mother. Finally. This study demotistl-dted lirnited clinical usefulness for these amniotic Huid solute determinations in this gro~rp of well-controllecl diabetic patienb. Small increases in maternal plasma glucose. amliiotic Ruid glucose, glycerol. and lactate and a larger increase irl amniotic fluid /K)H buryrate occurred in pregnancies resulting in neonatal I-espiratcq distress syndrome (Table IV) but were not statistically significant-. ‘l‘he lack of cot-relation of amniotic fluid solute levels to fetal h~lwglpcw~~ia or hypocalcemia most likel! also rcflectrd intensive management of these high-risk tteonates where immediate electrolyte and glucose parenter-al alimct~tatioti \vas used to reduce the incidence of’ these neonatal ha~ai-ds. 7‘his poor correlation 7vas not unexpcc ted in view of the relatively good metabolic control furnished these pregnant women and the small number of neonatal c-omplications. It is difficult to demonstrate a relationship between elevated levels of’ these amniotic fluid solutes ancl neonatal complications when there is a relativeI! small incidence of each oc‘l‘he authors are indebted to Dale Bucalo and Bernadette Plair for their t.echnical assistance and to Jane Holvroyde for statistical consultation.
Volume
135
Number 7
Changes in amniotic glucose, pOH butyrate, glycerol, and lactate 893
REFERENCES
1. Persson, B., ;i.nd Lunell, N. 0.: Metabolic control of diabetic pregnancy. Variations in plasma concentrations of glucose, free fatty acids, glycerol, ketone bodies, insulin, and human chorionic somatomammotropin during the last trimester, AM. J. OBSTE:T. GYNECOL. k!2:737, 1975. 2. Oaklev. , N. W.. Beard. R. W.. and Turner. R. C.: Effect of sustained maternal hyperglycemia on the fetus in normal and diabetic Ipregnancies, Br. Med. J. 1:466, 1972. 3. W iddas, W. F.: Transport mechanisms in the fetus. Br. Med. Bull. 17*107 1961. ., 4. James, E. J., Raye, J. R., Gresham, E., Makowski, E. L., Meschia, G., and Battaglia, F. D.: Fetal oxygen consumption, carbon dioxide production, and glucose uptake in a chronic sheep preparation, Pediatrics 50:361, 1972. 5. Drazancic, A., and Kuvacic, I.: Amniotic fluid glucose concentration, AM. J. OBSTET. GYNECOL. 120:40, 1974. 6. Wood, G. P.. ;and Sherline, I). M.: Amniotic fluid glucose: A maternal, fetal, and neonatal correlation, AM. J. OBSTET~ GYNECOL. 122:151, 1975. 7. Archimant, G., Gelizan, J. M., Ross, R. A., and Olthshe, 0.: Glucose concentration in amniotic fluid: Its possible significance in diabetic pregnancy, AM. J. OBSTET. GYNECOL. ll!k596, 1974. 8. Cassady, G., Blake, M., Bailey, P.. Younger, B., and Summers, J. Amniotic fluid glucose in pregnancies GYNECOL. complicated with diabetes, AM. J. OBSTET. 127:21, 1977. 9. Spellacy, W. N., Buhi, W. C., Bradley, B., and Holsinger, K. K.: Maternal, fetal and amniotic fluid levels of glucose, insulin and growth hormone, Obstet. Gynecol. 41:323, 1973. 10. Seeds, A. E.: Water dynamics in the amniotic Huid in Fair-weather, D. V. I., and Eskes, T, K. A. B., editors:
Discussion C. COODLIN, Omaha, Nebraska. Dr. Seeds is a recognized authority on the formation of amniotic fluid, and I have enjoyed and profited from reading his interesting paper. However, there are some features of the paper which I do not understand. Maternal ketones are considered to pass the placenta and to be a possible source of fuel for the fetus.’ I am puzzled by the authors’ statements that they “furnished evidence for the first time of ketoacids transfer across human placental tissue in vivo in diabetes.” Did the authors suspect that placental transfer would be different in diabetes or have they demonstrated a new observation that I missed? Dr. Seeds and colleagues believe that amniotic fluid solute concentrations reflect exchange with fetal circulation. I do no-t believe that anyone has proved this relationship to the exclusion of fetal membrane transfer. It is these membranes which presumably maintain the amniotic Huid composition of many constituents in cases of fetal d,eath. Several years ago, I thought we demonstrated that the human amnion may inHuence amniotic Huid Ielectrolyte composition. Human fetal membranes will function like a toad bladder by responding to oxytocin and by alterating sodium and chloride gradients.* I reasoned that the amniotic Huid DR.
ROBERT
Amniotic Huid-Research and Clinical Application, Amsterdam, 1972, Excerpta Medica. 11. Battaglia, F. C., and Meschia, G.: Principal substrates of fetal metabolism, Phvsiol. Rev. 58:499, 1978. 12. Kim, Y. J., and Felig, P.: Maternal and amniotic Huid substrate levels during caloric deprivation in human pregnancy, Metabolism. 21:507, 1972. 13. Paterson, P., Sheath, J., Taf‘t, P., and Wood, C.: Maternal and fetal ketone concentrations in plasma and urine, Lancet 1:X62, 1967. 14. Sabata, V., Wolf, H., and Lausmann, S.: The role of free fatty acids, glycerol, ketone bodies, and glucose in the energy metabolism of the mother and fetus during delivery, Btol. Neonate 13:7, 1968. 15. Smith. A. L., and Scar&n, J.; Amniotic fluid D (-) @-hydroxybutyrate and dysmature newborn infant, AM. 115:569, 1973. J. OBSTET. GYNECOL. 16. Felig, P., and Lynch, V.: Starvation in human pregnancy: Hypoglycemia, hypoinsulinemia, and hyperketonemia, Science 170:990, 1970. 17. Hoehorst, H. J.: L(+)-Lactate: Determination with lactic dehydrogenase and DPN, in Bergmeyer, editor: Methods in Enzymatic Analysis, New York, 1963, Academic Press, Inc., pp. 266-270. 18. W illiamson, D. H., Mellanhy, J., and Krebs, H. A.: Enzymatic determination of D (-) Beta-hydroxybutyric acid and aceto acetic acid in blood, Biochem. J. 82:90, 1962. 19 Pinter, J. K., Hayashi, J. A., and Watson, J. A.: Enzymic assay of glycerol, dihydroxyacetone and glyceroldehyde, Arch. Biochem. Biophys. 121:404, 1967. 20. Seeds, A. E., Leung, L., Clark, K., Stys, S., and Russel, P. T.: Permeability of chorion laeve in vitro to POH butyrate and glycerol. Suhmitted for publication.
of the anencephalic fetus might have electrolyte composition different from that of the normal fetus, since these fetuses often lack a pituitary gland. I have looked in vain for these changes in amniotic Huid electrolyte concentrations, but Pettit, King, and Blau” found the amniotic Huid of anencephalic fetuses to be very low in glucose. W e subsequently have analyzed the amniotic Huid of two anencephalic fetuses that were at 34 to 36 weeks’ gestation without severe hydraxnnios. In one, the fetus was alive at birth. In both cases, the amniotic Huid glucose was so low as not to be measurable, and in the live fetus its cord blood sugar was 36 mgi100 ml. Since fetal urine and tracheal Huid contain little or no glucose, I assume that fetal membranes must be a significant source of the amniotic Huid glucose. Amniotic Huid glucose concentration is known to decline in value with gestation, and to be very low in cases of chronic fetal distress and postmaturity. This decline perhaps reflects the increase in fetal swallowing and digestion described in these conditions,4 and its relative absence in the anencephalic fetus might indeed represent fetal hormonal effects on the amnion. Given the few exceptions of fetal disease as listed above, 1 think that we can assume that amniotic Huid glucose values, when corrected for gestation, reflect maternal blood sugar values. Since the authors’ paper gives LIS maternal data
894 Seeds et al.
onl! about fasting blood sugars, it is not possible lo conti1111e the argn1nent over whether the in viva fetus (or its membranes) is controlling the amniotic f-l&l levels of’ those solutes measured by the authors. Perhaps Dr. Seeds has more data on these solutes relativc to aninion transfer or maternal/fetal values. hly third question concerns Dr. Seeds’ assumption that 1he l’ctuses in this study all had increased insulin levels. (Znrrent dogma considers the fetus of the diabetic mother to have hyperinsulinemia,” but would this be true in those whose mothers are under the “good metabolic control” claimed 1~) the authors? ,Anlniocentesis is a commonly done procedure in high-risk clinics. I know that 1 have ordered all available laboratory tests on amniotic Huid in high-risk cases in hopes of finding an abnormalit\. that could have signilicance as far as fetal welfare is concerned. Howe\w, in the absence of si1nnltaneous fetal-maternal blooct studies, I think this sort 01’activity is unlikely to product significant acl~aricenicnt in fetology. There are exceptions, but most of out- useful laboratory testx of amniotic. ttuid constitnents were based on a prio1 deniotist~ation of their significant elevations in the concerned fetus. In m) opinion, Dr. Seeds a11d associates paper \\ould have more significance if siniultaneous fetal and maternal blood data were available f’or the solutes the\. 111easuld REFERENCES 1. 2.
3.
F&g, P.: Body fuel metabolism and diabetes in pregnancy. Med. Clin. North Am. 61:43. 1977. Goodlin, R. C., and Kresch, A. J.: Amniotic fluid osmolality following intra-amniotic injection of 23% saline, AM. J. OBSTET.GYNECOL.100:839, 1968. Yrttit, B. R.. King, G. S., and Blau, K.: Low glucose concentration in amniotic Huids from anencephalic pregnancy, Lancet 2: 1288, 1977.
‘1.
hlclain, C. R.: Amniography studies of the gastrointestinal motility of the human fetus, AM. J. OBSTET GYNECOL. 86: 1079. 1063.
I5
Adam, P. A.: Infant of a diabetic mother: Energy imbalance between adipose tissue and liver, Sem. Perinatol. 2.929 a. ., 197x.
DR. ALLAN B. WEINGOL~~, Washington, D. C. Dr. Seeds and his colleagues have addressed a most interesting and important subject in attempting to correlate amniotic Huid ketone levels with maternal glucose COIIcentration as a reflection of diabetic metabolic control. Current clinical practice calls for validation of fetal maturity prior to termination of a diabetic pregnancy. thereby providing us with one or ttiore sampies of arnniotic fluid prior to delivery. Utilization of this fetal solution f’w- predelivtiry alteration in therapy or for prediction of perinatal complications is a valid objective. The paper also serves to enhance our understanding of‘ the kinetics of ketone transfer across the placenta and draws the appropriate conclusions that meternal ketones are passed to the fetus and thus gain access to
tlie ami1iotic tluid. ‘I‘hc tlitIic.al illlpOlTilll~~C 01 Lchtosis Itt diabetic pregnant\‘. tiowt’wr, c~\-rcwds I,r\ontl tlw immediate association wirh feral cll~i1tl1i11 u1crc) 01. IICOnatal death. In 1069 Church,ll, Be~wtth al~tl Scnlorc.’ rcpxIing on the tle~elopment of infants of diabetic- mothers (1DN) from the I’erinatal (Mlaboratiw PIY+Y~I, 1101etl that the presenw ofa~~to1iu1-ia during pregnancy ws ;I critical variable itifluenciiig the intcltrctu.1l status of IDM’s at 4 years ot age. Inteltcctual inip3irnifnt \\as more often present in offspring whosc~ tiiotliei~s had displayed ;~cctoiinria, On the orticr I~i~tid. inlcllccti1al stat115 was unrelatecl to [Vhite’s classiticatioll, gcstational age, or the amount ot insulin rcwi\ctl I)\ the mother during prrgnanc! In 195’i. Stchbcns. Bakw. ;111d Kitchill” c onfirtnetl these results in a prosptt.tiw sruti\ on an rnlirelv different popiilatioti. .l‘tica (Iif’trcnce bct\vwti IQ scow at .5 years of age 01’children born to aceto11c-iieaati\e is. ;icetone-l,ositi\.e morhcrs \+.assignificanr at p < 0.05. It is important to nolc that 1he caust’ 01‘awtot1~lria \\‘a> not specified in eittrcr stIlti>. It ina? haw rc~prcsentcd true diabctit kctoacidosis scw~ntlar~ to poor control (insiifficicnt insulin or cxc es5 calories) or thr pregnatic\ rnhariced tcntlrnc-\ to starvation ketosis sccontl;~r~ to insufficient calories. I’hc critical itnportaticc ot adcquate niaternat caloric inrake in diahetit pregnanc\~ with entphasis 011 coniples ~a1boh~dratcs ~arrnot be ovcret11l)liasi~~~l.” For exi1nlple, nlater11al bt~~~~tllevels ot beta-l1~dr~~x~buqrate and acetoacetic. acid are tl\o 10 f’our times greater in pregnant>’ al‘tc.1.o\crnight fasting than in the nonpregnant state.’ Maternal ketonemia is associated with ketone body acc11mulation in the amniotic flnid and, bp inference, s11ggesfs that the fc*tus is exposed to substances that tuav ha\~ an atl~erae et’l’ec~~on subsequent 11twr&eha\ ioral de\ clopment. I‘ptake trf ketone ac.ids has her11 observed in the human fetus and the enzymes f’or ketone oxidation have bee11 idcntilied in fetal brain tissne.” Perhaps 1his is tl1e major problem with Dr. Seed’s syndy. it ma) be a USC OF wrong time at1d wrong place. Talented physicians. managing overt diabetes in pregnancy, who generally achieve their objective of eugl~cemia, are simpty not going to see significant l~laternal ketoacidosis. Nevcrt helcss, how much mow inform+ tive this paper ww1ld ha\ e hecn if’ maternal ketones had been studied and paired \\+th amniotic fiuid lwels. A comparison of maternal filu~oseihydrox~b~~t~r~1t~ levels VS. amniotic levels of the same componnds ~onld have frilly excluded a starwtion ketosis cwnptment and may Ivet have clarilicd rhe var>’ large standard dcviation disptaycd in the beta-hvtl1-o?cybutyrate levels. TM) first question, then, of Dr. Seeds is “Cl’as there a specific reason ~rhy maternal levels of1he solutes stndied, other than glucose, were not rneasurecl?” A second cIuestio1i relates to another substance which Inay reflect glucose metabolism a11d has brcrl (‘1 altla1Nl
Volume 135 Number 7
Changes in amniotic glucose, p0l-i butyrate, glycerol, and lactate 895
by amniotic fluid analysis, namely, insulin. Amniotic fluid insulin appears to be totally fetal in origin as evidenced by the failure to measure 112”labeled insulin in that compartment after maternal infusion. Levels of greate;. than 60 FLU/ml, particularly in association with low amniotic Iluid levels ol‘ glucose, correlate wel! with poor perinatal outcome. ’ Dr. Seeds, could your studies be extended to include insulin assessment? Do you believe that amniotic ketones are a IIKX~ accurate “memory” statement about pre-existing metabolic control than insulin? REFERENCES
Churchill, J. A., Berendes, H. W.. and Nemore, J.: AM. J. ~BS1.E’~. GYPIECOL. 105:257, 1979. Stehbens, J. A., Baker, G. L., and Kitchell, N.: AM. J. ORSTET. GYNECOL. 127:408, 1977. Weingold, A. B.: Diabetes in pregnancy, pg. 163 in Caplan, R. M., and Sweeney, W. J., editors: Advances in.Obstet and Gynecol, Baltimore, 1978. The Williams & Wilkins Company. 4. Felig, P., and Lynch, V.: Science 170:990, 1970. 5. Page. M. A.. and Williamson, D. H.: Lancet 2:66, 197 1. 6. Newman, R., and Tutera, G.: Obstet. Gynecol. 47:599, 1976. DR. SEEDS (Closing). Dr. Goodlin’s series of questions reflect his understanding and expertise in this field, and his innovative thinking about this area. His statement about the lack of significance I would like to modify to make clear that although limited clinical usefulness of this study was demonstrated, there was a clear statistically significant relationship demonstrated between increases in amniotic Huid, betahyclroxybut~ratc, and increases in maternal serum glucose. T-his strong relationship provided firm evidence in the human subject that beta-hydroxybutyrate is crossing the placenta from the mother since the fetus would not be producing this compound in excessive amounts in the presence of high insulin levels. Moving on to some of the other points that Dr. Goodlin brought up, and they are all interrelated, my under-standing of amniotic fluid formation is pure and simple. It is largely a product of exchanges with the fetus and incleascs in solutes in amniotic fluid, with very few exceptions, very likely parallel increases of the same solute, in the fetus. If solute concentrations do not increase in the fetus, they do not get into the amniotic fluid. For this reason I think this kind of evidence, i.e., amniotic fluid increase in beta-hydroxybutyrate, as well as glucose, reHects maternal transfer of these solutes across the placenta to the fetus. I was as surprised as Dr. Goodlin, having read a number of textbook statements implying that, of course, ketoacids cross the placenta, and accepting this as fact and mechanism, for the great threat to th,e fetus represented by maternal acidosis in diabetes. However, extensive review of the literature failed to provide evidence supporting this concept. Theoretical reasoning
suggests that ketoacids might not cross the placenta since they have a low pK (3 to 4) and are largely ionized at physiologic pH. Because of such physical chemical characteristics, these compounds could be expected to cross placental tissue with difficulty. Now, looking at evidence in the literature for the transfer of these solutes from mother to fetus, in the sheep they do not cross at all. In the human subject it seems very clear to me that all available data indicating transfer were obtained where the mother was fasted and the fetus then hypoglycemic and hypoinsulinemic. Tht fetus could thus be producing these ketoacids and not accumulating them by placental transfer from the mother. Under these conditions, small parallel rises in maternal and fetal ketoacids do not provide strong evidence of placental transfer. I looked for data on cord blood or amniotic fluid increases in ketoacids during the diabetic pregnancy, where the hyperglycemic hyperinsulinemic fetus would not be a primary source of ketoacids, and there is limited or zero evidence in that area, and hence this study. It is my contention that this study then for the first time provides evidence in pregnant diabetic subjects of maternal to fetal ketoacid transfer. Clearly this study would be stronger if simultaneous maternal serum ketoacids had been measured, as both Drs. Goodlin and Weingold suggested. One reason why this was not done is that about half of this study was retrospective. ive had the fluid collected in these cases before planning these analyses. A prospective study is now under way in which maternal serum and amniotic fluid ketoacid glycerol and free fatty acids and amniotic fluid insulin, glucsgon, and C-peptide are being measured. We will try to answer these kinds of questions. Dr. Goodlin mentioned fetal membrane function as possibly similar to toad bladder, and cited experiments with oxytocin, where in vitro permeability of placental membranes was apparently changed. Such changes might influence amniotic fluid values and we cannot exclude that possibility. However, in our laboratory, 5 or 6 years ago, we did study the effect of oxytocin vasopressin and 3’5’AMP on placental membranes to see if there was an effect similar to that found with toad bladder where pore size and membrane permeability are affected and there is increased permeability to solutes and water. In a large over a \vide concentration number of experiments, range of these agents, we found no in vitro response or change of in vitro placental permeability in human subjects. Finally, regarding Dr. Weingold’s excellent remarks, I agree that amniotic Huid insulin measurements yould improve the study. Amniotic fuid insulin is probably of fetal origin and may well provide a better memory system then ketoacids. For this reason our prospective study includes measurements of insulin, C-peptide, and glucagon.
