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COMPOSITION OF ALVEOLAR LIQUID IN THE FOETAL LAMB
BY T. M. ADAMSON, R. D. H. BOYD, H. S. PLATT AND L. B. STRANG From the Department of Paediatrics, University College Hospital Medical School, London, W.C. 1
(Received 24 February 1969)
1. Experiments were performed on foetal lambs at gestations between 125 days and term. The foetus was exteriorized at Caesarean section with the umbilical cord and placental attachment maintained intact. Samples of liquid from the alveolar parts of the lung were withdrawn through a tracheal cannula and samples of lung lymph, plasma and amniotic liquid were also obtained. Measurements were made of total osmolality, concentrations of electrolytes and urea, pH and Pco2. Titrations were carried out with N/10 Hal and N/10 NaOH. The water content of the liquids was estimated and concentrations expressed per kg H20. 2. In alveolar liquid [H+], [K+] and [CO-] were higher and [Ca2+], [phosphates] and [HC03-] were lower than in plasma or lymph. In amniotic liquid osmolality [Na+], [Cl-] and [Ca2+] were lower and [phosphates] higher than in plasma or lymph. Alveolar liquid/plasma ratios of [HCO3-], [Ca2+], [CI-] and [K+] differed from ultra filtrate/plasma ratios of these ions. 3. Titration curves demonstrated a very small amount of buffering in alveolar liquid at its in vivo pH of 6-27 mostly due to HCO3- at an average concentration of 2-8 mM/kg H20. 4. It is concluded that foetal alveolar liquid is not an ultrafiltrate of plasma nor a mixture of amniotic liquid and plasma ultrafiltrate, but a special material elaborated by the foetal lung.
The lungs of foetal mammals including humans are filled with liquid (Addison & Howe, 1913; Potter & Bohlender, 1941; Adams, Moss & Fagan, 1963). In the lamb at term the volume which can be aspirated through the trachea is about 25 ml./kg body wt. (Humphreys, Normand, Reynolds & Strang, 1967). Since ligation of the trachea in the rabbit (Jost & Policard,
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D. 2011 . alveolar liquid was sampled by cannulating the trachea. Hastings & Neill. it is very likely that the liquid is formed in situ. 1941) leads to overdistension of the foetal lungs.160 T. and the buffering in alveolar liquid differ from plasma and from lung lymph. Carmel. in an Autoanalyser (Technicon) by the method given in the Technicon Handbook (file N-21a). after equilibration with 5% C02. This had an S. Titration curves were obtained by titration in room air with N/10 HC1 and N/10 NaOH. but in nine experiments halothane was given to the ewe by inhalation instead of pentobarbitone. range 1-87-5-7 kg). ADAMSON AND OTHERS 1948. for five repeated measurements on the same sample. M. METHODS Experimental procedure Experiments were performed on forty exteriorized foetal lambs with gestational ages between 115 days and term (mean weight 3-9 kg. amniotic liquid and whole blood collected anaerobically by syringe. of repeated measurements < 1mm/1. Alveolar liquid has a low concentration of protein (0.D. in carotid artery blood. and neither a simple ultrafiltrate nor a mixture of amniotic liquid and ultrafiltrate. Analysis of liquids Acid-base measurements. 1967).). of + 0-23 mM/l. (1967). 1965) or congenital bronchial atresia in humans (Potter & Bohlender. and gently aspirating the remainder anaerobically into a syringe.. These measurements suggest that alveolar liquid is a special material elaborated by the foetal lung. [HC03-] was calculated by subtracting dissolved CO2 plus H2C03. Boston. The much higher concentrations of total CO2 in lymph. a sample of amniotic liquid taken anaerobically into a syringe. Friedman & Adams. 1967). Humphreys. and the foetus delivered onto a heated table with the umbilical cord and placental attachment intact. Sendroy.03 g/100 ml. clearing the dead space by withdrawing and discarding between 10 and 30 ml.physoc. (1963) reported that the liquid differs strikingly from plasma in its low total CO2 content and pH. The thoracic duct component of lung lymph was collected as described by Humphreys et al. with results similar to values previously published (Humphreys et al. many of which were used also for other experiments. But Adams et al. 1968) which suggests that it could be formed by ultrafiltration from lung capillaries. the pH of lymph was measured in samples collected under oil. which we have taken to represent interstitial fluid. Reynolds & Strang. plasma and amniotic liquid were measured. The foetuses were shown to be in good condition by monitoring P °02 P0Co2 and pH. A Caesarean section was performed. We have also compared alveolar and amniotic liquids. liquid. Downloaded from J Physiol (jp. and samples of heparinized carotid artery blood were collected anaerobically. The measurements reported in this present paper were made to establish how much the concentrations of substances. Total CO2 was measured in uncentrifuged anaerobically collected samples of alveolar liquid by the Natelson modification (1951) of the Van Slyke method (1917).org) by guest on November 16. (S. In thirty-one experiments anaesthesia in the ewes was induced with thiopentone and maintained with pentobarbitone as previously reported (Humphreys et al. 1928). Measurements of Pcol by the method of Severinghaus & Bradley (1958) and of pH using a capillary glass electrode were made in samples of alveolar liquid. estimated as 0-03 x PCO2 (Van Slyke. Normand.
No differences in concentration were observed depending on how much liquid was aspirated from the trachea before taking a sample.s. Osmolality was measured by freezing point depression (Advanced Instruments osmometer). centrifuged at 30. [HCO3j-. and from Humphreys et al. No differences related to gestational age were observed. one taken after withdrawing 3 ml. (1967) for lung lymph and plasma.05). These estimates agreed to less than 0. these compare with Davson's (1956) value of 990 g/l. The measurements were made on samples of alveolar and amniotic liquid. Figure 1 compares ultrafiltrate of plasma/plasma ratios (Rufp) given by Davspn (1956) with alveolar liquid/plasma ratios (Raiip) from measurements on simultaneously collected pairs of samples from seven animals. colourless but more opalescent on collection. [K+]. In plasma the sum of measured anions (excluding phosphates) is 25 m-equiv/kg H20 less than the sum of cations. amniotic liquid. the difference is presumably made up by anionic protein. for c. respectively were obtained by evaporating to dryness. 6 Phy. Alveolar liquid. (1968) for alveolar liquid. Thus Table 1 probably includes all the quantitatively important anions in alveolar liquid. Concentrations of Na+. which has a similar protein concentration to alveolar liquid. For alveolar liquid and amniotic liquid.2% with measurements made by evaporating to dryness. [Na+]. electrolyte and urea concentrations and for pH and PC02 in alveolar liquid.000 g and 00 C for 10 min. Measurements were also made on six samples of uncentrifuged alveolar liquid and showed no important difference from results after centrifugation.org) by guest on November 16. [C1-] and [HC03-] differed by less than 1 mM/kg H20 and pH by 0-02. Alveolar liquid and plasma. [Ca2+] and [phosphate] are significantly lower than in plasma while [Cl-]. On centrifugation amniotic liquid. and in one particular experiment in a pair of samples. retained its colour and some opalescence.. [protein].f. 2011 . The Table includes values for protein concentration from Boston et al. The coefficients of variation of repeated measurements were 1% or less. 1954). organic acids and phosphates (Gamble. became crystal clear on centrifugation. for lymph were obtained from the equation of Peters (1953) relating serum protein and H20 concentrations. water concentrations of 990 and 986 g/l. 204 Downloaded from J Physiol (jp. In alveolar liquid. and the other after 30 ml. N-4b and N-ic) and Ca2+ by the method of Halse (1968). lung lymph and plasma.ALVEOLAR LIQUID COMPOSITION 161 Measurement of electrolyte and urea concentrations. [K+] and [H+] are significantly higher (P of the above differences < 0-001 except for [K] where P < 0. RESULTS Concentration of &ubstances Table 1 gives a mean values for osmolality.physoc. In alveolar liquid the sums of anions and cations are similar (anions 2-6 m-equiv/kg H20 greater than cations). pale yellow and faintly opalescent on collection. For this purpose average water concentrations of 956 g/l. K+ Cl-. Concentrations are expressed per kg H20. urea and phosphates (as P) were made using an Autoanalyser (Technicon) by methods given in the Technicon Handbook (files N-21a. for plasma and 960 g/l.
Cf .t -III5 _eN ) = (=> CO aq o 00o t O r to ° 10 (M C) 1- P-Z C) ao CD - Co aq 0 cs0 r to 4* CO-d M CO o * * .o aq oo5 06 c.org) by guest on November 16.e aO> W .Peq C>0-4 co oq - 1o >.162 T. q0 CO O 00 Co 0 C o C4 - - I I0 --I M M It 0 0 0 c cl_ so~ . C or= .4 . 2011 . CM 01 o-- - * aq co 1 C O r CO tZ. O I 0 * . ADAMSON AND OTHERS .c c 0 9d 0 0 Co V V CO -1 to t L IN I- I C. M. -= ..- V _ *-Xr O- ?_^-~ M _1 (M 1:H O X r- . °: Co 0A o -no 0) COO 0COc 10 I OCOO ..5 6 0 1 1-: * o 0o 00- rq C 0 to +.6 _= M:.~o b.C bo -0C o0 M Z O t- _o0 * -0 - 0 S- 32 _ ~~~~~~~~~~~C) ce ol > :3 rg 0o j . C4E0r.0*> o c) o 3 + 0 C) + 0C H M o 1 Co 0 CO10 Mo 01:.- coE Downloaded from J Physiol (jp.physoc.
e.. open columns) ratios of concentration (mM/kg H20) with + s. In amniotic liquid [Na+].. HC03.1:. filled columns) and alveolar liquid/plasma (Raiip.-.'.-_.' .:w :>:::: i::..E. and from the S.:-.. Rasp ratios from simultaneously collected pairs of samples in seven foetal lambs..physoc. There were no significant differences between lymph and plasma in the concentrations of the other substances measured in paired samples. and for [C1-] and [K+] are higher than the corresponding Ruvp values.S given by Davson (1956) the differences appear to be significant. [phosphate] and pH higher (P < 0-001) than in alveolar liquid.. Titration curves Figure 2 shows typical titration curves between pH 4 and pH 10 for alveolar liquid.|. A. Mean ultrafiltrate/plasma (Rflp. water and a solution of NaHCO3 (4 mm/l.'.. shown where available as vertical bar. [Cl-] and osmolality are lower (P < 0-001) and [HC03-]. 0 cICast NaO Osm.ofn-. 2011 . '. Rudp ratios from Davson (1956). The slope of the titration curve for alveolar liquid is much less steep than for lymph or amniotic liquid and indicates a small amount of 6-2 Downloaded from J Physiol (jp.: 'm-:'::':' >er^Z Amniotic liquid.:. In A:. 1-6 r- 1-411-2 11-0 I- F' 0-810610-41-- 0-21-.). .: >.E. amniotic liquid. ::. e> T :.-:': :'' . 1.:.'. lymph....Urea Fig. Lymph..' He . - '. . There is a lower protein concentration in lymph than in plasma.163 ALVEOLAR LIQUID COMPOSITION The Rai/p values for [HC03-] and [Ca2+] are substantially lower...org) by guest on November 16. [Ca2+].
alveolar liquid (e). 2 (Van Slyke.-l pH-') (Van Slyke. and at a pK.T. 164 . This relationship is illustrated in Fig. Figure 3 gives buffer values (mm[H+] per litre and unit pH change) obtained from the slopes of the curves in Fig. The actual position of the titration curve for lung liquid on the ordinate depended on the Pco. amniotic liquid (L). and NaHCO3 solution. 1922). Titration curves of 5 ml. Ordinate: mM-HCl and mm-NaOH added. lung lymph (0). (M). at the start of the titration. 4 5 6 In a solution of a single buffer. but differences in Pco. buffer concentration (mM/l.0-04 _° 0*03 _ 0 o e.physoc.org) by guest on November 16. M. Amniotic z 0-02 E: _ 0. water (A). pK values are given by points of maximum slope. which was not controlled.01 Water 0 _ 0D01 S IL £ 0*02 _- Lymph 003 _ 10 8 9 7 pH Fig.) x 0575 = buffer value (mm[H+]l. solution of NaHCO3 has a buffer value of 2 3 at its pK. The NaHCO3 solution gave a Downloaded from J Physiol (jp. do not affect the slopes of the curve. 4mM/I. 1922). 2. ADAMSON AND OTHERS buffering in this liquid. Abscissa: pH. 3 where the 4 mM/l. 2011 .
) mM/kg H20.org) by guest on November 16.ALVEOLAR LIQUID COMPOSITION 165 standard buffer curve with an expected pK at 6x37. to amino acids with pK values mostly between 8 and 12. The buffering additional to bicarbonate in the low pH range is likely to be due to intermediary metabolites (e. At the pK of HC03. The closeness of the peak in buffer value of alveolar liquid to the pK of aqueous HC03solution suggests that most of the buffering in alveolar liquid at this pH is due to its HC03. pH-'.g.this would give a mean buffer value of 1-5 (± 0. indicating the presence of small amounts of additional buffer in the low pH range and relatively large amounts in the upper part of the range. 2011 . 2 of alveolar liquid (0). and in the upper pH range 8 7> Alveolar_ 6 I 0. Ordinate: buffer value mm H+ . Abscissa: pH.) (M).l. Buffer values obtained from the slopes of curves shown in Fig. which compares with a measured mean buffer value of Downloaded from J Physiol (jp. The curve for alveolar liquid is similar to that for NaHCO3 solution but differs slightly in shape from the standard curve. -1 I i4 E 0 >3 2 1 Water 0 _ A `-I- - - - - - - - h - - - H - I_# 4 5 6 7 8 9 10 pH Fig.2) mm H+. water (A) and NaHCO3 (4 mm/l.-i.E. The mean [HC03-j in alveolar liquids where titration curves were performed was 2-7 (± 04 s.physoc. 3.pH-1.content. lactic acid and Krebs cycle acids) with pK values below 6. 1 .
such as those published by Weibel (1963). while there are pores or clefts between pulmonary capillary endothelial cells.-'. variable Downloaded from J Physiol (jp.L. By expressing the measured concentrations in alveolar liquid per kg H20 and comparing them with Davson's (1956) values for plasma ultrafiltrate. Normand.physoc. which has an electrolyte composition similar to plasma.166 T. the whole cellular lining of the lung must be capable of maintaining the observed electrolyte concentration differences from plasma. H+. the junctions between alveolar cells appear to be tight. show distinct capillary endothelial and alveolar epithelial cell layers. Thus in alveolar liquid at its in vivo pH there is a small amount of buffering (average buffer value 0. but since the liquid was not thoroughly equilibrated with air before starting the titration. and which has been shown by Boyd. The results given in Table 1 show that alveolar liquid in the foetal lamb has an electrolyte composition which is quite different from either amniotic liquid or carotid artery plasma. Humphreys. Schneeberger-Keeley & Karnowsky (1968) have shown that. Plainly the largest area across which the concentration gradients are maintained consists of alveolar walls. because [Ca2+]. Hill. M. Reynolds & Strang (1969) to have the macromolecular composition expected from the filtration of plasma through porous capillary walls. Since no variations in composition were found depending on how much liquid was aspirated before taking the sample. Indeed it seems likely that alveolar liquid is a special material actively elaborated by the foetal lung in contrast to lung lymph.org) by guest on November 16. pH-' at the pK of HCO-. The present experiments provide no evidence as to the part of lung in which the liquid is formed. 2011 .7) in addition to HCO3-. The titration curves were performed in air at an effectively zero PCo2. Electron micrographs of lung. ADAMSON AND OTHERS 2X2 (± 0-2) mM. The mixing together of amniotic with a plasma filtrate formed in the lungs could not account for the composition of alveolar liquid. DISCUSSION Electrolyte composition of alveolar liquid. we have shown that the differences in ionic concentrations of alveolar liquid from plasma are not simply imposed by the Donnan equilibrium as a result of the difference in protein concentration. The low protein concentration and special electrolyte composition of alveolar liquid would appear to depend on the properties of the alveolar cell layer. [phosphate] and [HCO3-] are lower and [Cl-] and [H+] higher in alveolar liquid than in either of the other two liquids. Acid base composition of alveolar liquid. which in the foetus is likely to differ only slightly from pulmonary artery plasma due to a contribution which has passed through the lungs.
(1952). K. If the active addition of acid to alveolar liquid is the reason for its low [HCO3-]. B. Biologia Neonat. p. W. & ADAMS. C.167 ALVEOLAR LIQUID COMPOSITION amounts of dissolved C02 would be present in the liquid. R. CARMEL. HALSE. FRIEDMAN. NORMAND. Anat.. 151-158. H. F. F. E. 1962) and contains carbonic anhydrase (Berfemstam. 452-456. & STRANG. (1963). Moss. R. & FAGAN. London: Churchill. 2011 . D. (1956). 41. S. Liberation of zinc by calcium from Zn EGTA in the presence of a zinc indicator. It is possible that alveolar liquid is secreted by a process similar to HCl secretion inl the stomach.. I. On the prenatal and neonatal lung. 199-214. of 40 mm Hg. The titration curves show that most of the effective buffering in alveolar liquid at its in vivo pH is provided by its small concentration of HCO3-. (1968). Physiol. Am. A. (1948). Neonat. C. Physiology of the Ocular and Cerebral Fluid. HUMPHREYS. 567-588.. W. DAVSON. ADDISON. Am. J. would be unaffected by these differences in [C02]. J. 310-315. This work was supported by a grant from the Medical Research Council in laboratories constructed with aid of a grant from the Wellcome Trust. 1952). ADAMS. 1948). R. 306-35. H. Automated photometric estimation of blood calcium. A. 0. 5. E. Child. HUMPHREYS. A. Hydrochloric acid production by isolated gastric mucosa. New York: Medical. Acta paediat. R. (1954). 5. REYNOLDS. R. 6th edn. 109. which would affect the pH at the time of starting the titration and hence the position of the titration curve on the ordinate. p.. (1969). L. I. 0. GAMBLE. P. which were therefore considered unimportant. 143-149. H. The tracheal fluid in the fetal lamb. REFERENCES F. J. Dis. NORMAND. 12.. Carbonic anhydrase in foetal organs. Fetal tracheal ligation and lung development. L. BOSTON. J. (1913). II. (1968).. J. which has been shown to depend on transfer of H+ from H2CO3. vol. The low [HCO3-] could be due either to the active exclusion of this substance or to the active addition of a strong acid. J. Formation of liquid in the lungs of the foetal lamb. Stockh. W... Cambridge. R. Technicon Symposium 1967. Mass: Harvard. pp. R. DAVIES. Biochem. 201. L. 15. REYNOLDS. In Automation in Analytical Chemistry. and to require carbonic anhydrase (Davies. Boyd & Mossman. 67. F. and hence the estimate of buffering.. H. Chemical Anatomy. Downloaded from J Physiol (jp. H. H. HILL. W.org) by guest on November 16. J. Physiology and Pathology of Extracellular Fluid. Biologia. P. 42. B. (1965). which would lower [HCO3-] by releasing C02 with its diffusion into plasma.. J.physoc. BERFEMSTAM. & STRANG. & HowE.. BOYD. W. S. The pH of 6-27 can be explained by equilibration of this poorly buffered liquid with a PCO. 609-618. the acid is probably HCl as any other would be represented as a deficit in measured anions compared with cations. L. But the slope of the curve at any pH. Permeability of lung capillaries to macromolecules in foetal and new-born lambs and sheep. The foetal lung is derived from foregut (Hamilton.
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