MICROVASCULAk RESEARCH 50, 373--380 (1995)

Age-Related Changes in Albumin-Excluded Volume Fraction

SANDHYA PARAMESWARAN, B. J. BARBER, R. A. BABBITT, AND S. DUTTA

Center for Biomedical Engineering, University of Kentucky, Lexington, Kentucky 40506-0070

Received September 16, 1994

The space available to large macromolecules, such as albumin and globulin, is less than the total interstitial fluid volume due to the dense matrix formed by the interstitial ground substance. Changes in excluded volume are likely to indicate changes in the composition of the matrix. Sprague-Dawley rats were anesthetized with sodium pentobarbital. Serum, mesenteric tissue, and peritoneal fluid samples were obtained. Albumin contents were determined by microrod electrophoresis. Serum and mesenteric tissue chloride concentrations were measured by the coul, ometric-amperometric method. Serum and mesenteric tissue sample chloride concentrations were not significantly different, suggesting that this loose connective tissue is composed almost entirely of extracellular matrix. Matrix hydration decreased with a regression slope of -0.014 (#g tissue water/#g tissue dry wt)/10 days. Serum and tissue albumin concentrations decreased between 210 and 630 days of age. Mesenteric loose connective tissue albumin-excluded volume fraction increased by 80% over this age range. The increase could not be accounted for by dehydration alone, suggesting that the increase in excluded volume fraction for albumin is also due to changes in tissue glycosaminoglycans or collagen. © 1995 Academic Press, Inc.

I N T R O D U C T I O N

The g lycosaminoglycans and col lagens in the intersti t ial ground substance form a dense matr ix making certain regions of the meshwork inaccessible to solute species. A~ a consequence, the space avai lable to large macromolecules , such as a lbumin and globulin, is cons iderab ly less than the total interst i t ial fluid volume. Exc luded volume for a given solute depends on the size o f the solute and the concentrat ions o f the various components o f the matr ix (Ogston et al., 1973). Changes in exc luded volume are l ikely to indicate changes in the composi t ion of the matrix. Protein osmotic act ivi ty is enhanced in the interst i t ium as a result o f p lasma protein exclusion (Granger et al., 1984). Albumin is responsible for a major part of the col loid osmot ic pressure in p lasma and intersti t ial fluid. Previous studies have shown that a lbumin is excluded from a substantial part of many intersti t ia (Bert and Pearce, 1984).

Col lagen mass is concentrated into rods while g lycosaminog lycan mass is d ispersed over a three-dimensional volume due to random coil ing of the long mucopolysaccha- r ide chain. Therefore, g lycosaminoglycans occupy a much larger solvent vo lume than col lagens (Granger, 1981). Tissue g lycosaminoglycans are altered in aging (Vogel , 1980). Changes in hyaluronic acid concentrat ion would alter the exc luded volume fraction and reflection coefficient for a lbumin; this in turn determines the fluid and protein distr ibution between the vascular and the extravascular spaces (Granger, 1981). However , because of the larger concentrat ion of col lagen in certain t issues and dermis,

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3 7 4 PARAMESWARAN ET AL.

collagen may also play a major role in plasma protein exclusion (Wiig et al., 1992). Tissue collagens change with age (Kohn and Schnider, 1989). Cross-linking tends to decrease the ratio of soluble to insoluble collagen. Increased collagen cross-linking would tend to increase the plasma protein-excluded volume fraction.

The purpose of the present study was to measure the changes in albumin-excluded volume fraction with age. The rat mesenteric tissue drape was chosen because this model has been widely used for microvascular studies (Gore, 1986) and has avascular regions suitable for microchemical analytical techniques. Mesenteric tissue and serum chloride concentrations were measured to determine the tissue extracellular matrix fraction. Age groups were chosen to correspond to juvenile, adult, midlife, and old; 10 days of aging in a rat is approximately equivalent to 1 year in humans (Lowry and Hastings, 1952).

MATERIALS AND METHODS

Male Sprague-Dawley rats (age 80 days, n = 13; 210 days, n = 18; 390 days, n = 11; 630 days, n = 22) were fasted overnight, but allowed water ad libitum. The animals were anesthetized by intramuscular injection using sodium pentobarbital (65 mg/kg body wt) and ketamine (50 mg/kg body wt). This avoids any possibility of diluting the peritoneal fluid with an intraperitoneal injection. A heated board was used to maintain colonic temperature at 37-38 ° (Yellow Springs Instruments, Model 46TA, Yellow Springs, OH) and the temperature was measured using a digital rectal probe (Yellow Springs Instrument Co., Model 49TA). The femoral artery was catheterized with PE50 tubing for blood pressure monitoring. A 4-cm midline incision was made caudally on the ventral surface of the abdomen starting at the xiphoid process using heat cautery to prevent bleeding and seepage into the gut. An ileal intestinal loop was exteriorized to expose the mesenteric tissue.

A 200-#1 blood sample was obtained immediately after the femoral catheter was inserted. Hematocrit levels were measured with a microcapillary reader (Damon/IEC Division, Model 2201). The remaining sample was centrifuged and the plasma was stored in a refrigerator. Peritoneal fluid samples were collected by pipetting (Rainin Instrument Co., Inc., Woburn, MA) from within the folded intestinal loops.

Mesenteric tissue samples were taken as previously described (Barber et al., 1990). Briefly, an ileal intestinal loop was rapidly exteriorized and the mesentery was draped onto a supporting block covered with parafilm. A fat-free avascular region was selected and a tissue button was punched eCut using an 8-mm circular trephine (Weck Type 1703, Research Triangle Park, NC). Tissue dry weights and water content were obtained by microgravimetric analysis (Barber et al., 1993). Plasma, peritoneal fluid, and tissue albumin content were determined'by microrod electrophoresis (Barber et aL, 1990). Total protein content was determined by the Lowry method (Sigma 690-A).

The excluded volume fraction for albumin was calculated from the mesenteric tissue and peritoneal fluid albumin concentrations as (Barber et al., 1990)

c~ f ~ = l - m Cp'

wherefe = albumin-excluded volume fraction, Ct = mesenteric tissue albumin concen- tration, and Cp = peritoneal fluid albumin concentration.

EXCLUDED VOLUME 375

This assumes that mesenteric tissue is bathed in peritoneal fluid and hence is in equilibrium with the fluid.

The chloride concentrations of 20-#1 serum and tissue samples of 630-day-old rats were measured in meq/liter by a coulometric-amperometric method using a chloride meter (Coming Model 920M). To obtain a detectable concentration of chloride, 8 to 10 tissue buttons were pooled together and reconstituted with 60 #1 of sample buffer (0.2 g sodium dodecyl sulfate, 1 ml glycerol, 7.8 ml water, 1 ml stacking gel buffer, pH to 6.8 with HC1). The conditions for preparing the samples were the same as for microrod electrophoresis.

An analysis of variance was performed with the treatment groups consisting of the different age groups. Age-related trends were tested for by linear regression analysis. Analysis of variance (ANOVA) and regression analysis were performed using a statis- tical software package (Systat, Evanston, IL). Bartlett's test was used to test for homogeneity of variances. A significant Bartlett test result implied that ANOVA was of questionable validity; in this case, a Kruskal-Wallis nonparametric multiple comparison test was used to test for significance between age groups.

Results are expressed as means _+ standard error, unless otherwise mentioned. Values are assumed to be significant at the P < 0.05 level.

RESULTS

Tissue water (#g) was calculated as the difference between total wet weight (#g) and dry weight (#g). Mesenteric tissue sample hydration was calculated as a ratio of tissue water (#g) to tissue dry weight (#g). Tissue wet weight and dry weight increased with age (Table 1). Significant differences in hydration were found between age groups (ANOVA, P < 0.0001) and the changes were correlated with age (r = -0.7). Regression analysis of the decline in hydration resulted in a significant (P < 0.0001) sk~pe of -0 .014 _+ 0.002 (#g tissue water/#g tissue dry wt) per 10 days.

Serum total protein increased progressively with age (see Table 1). Significant differences were present between age groups (ANOVA, P < 0.0001). Serum total protein was positively correlated with age (r = 0.6). A significant (P < 0.0001) positive slope of regression was obtained, 0.014 _+ 0.002 (g/dl) per 10 days. However, it should be noted that the change between 210 and 390 days of age appears to be less than the trend while the change from 390 to 630 days of age exceeds it. Unlike serum total protein, tissue total protein concentration decreased with age (see Table 1). Significant differences were pre~ent between age groups (Kruskal-Wallis test, P < 0.0002). Tissue total protein was negatively correlated with age (r = -0.6) . Trend analysis gave a significant (P < 0.0001) slope of -0 .014 _+ 0.002 (g/dl) per 10 days.

Serum albumin concentration exhibited nonlinear behavior with an initial increase from 80- to 210-day-old rats and a decline thereafter. There were significant (ANOVA, P < 0.0001) differences in serum albumin concentration between age groups, and the changes were correlated with age (r = -0 .7) from 210 to 630 days of age. Regression analysis on the data from the three age groups (210-630 days) indicated a significant trend (P < 0.0001) with a slope of -0 .02 _+ 0.002 g/dl per 10 days. Unlike serum albumin, mesenteric tissue albumin decreased progressively with age. Significant dif- ferences were found between age groups (ANOVA, P < 0.0001). Tissue albumin was negatively correlated with age (r = -0.7) . A significant (P < 0.0001) slope of regression, -0 .009 _+ 0.001 (g/dl) per 10 days was obtained. Albumin concentration

376 PARAMESWARAN ET AL.

TABLE 1 Age-Related Contrast of the Means and Tukey HSD Multiple Comparisons

for Various Physiological Parameters

80 day 210 day 390 day 630 day

Serum protein (Lowry) 5.5 ± 0.1 5.8 ± 0.1 6.0 ± 0.2 6.4 ± 0.081"** (g/dl) (4.6-6.4), 13 (4.9-6.7), 19 (5.1-6.9), 10 (5.8-7.1), 20

Albumin 1.9 ± 0.07 ~,*** 2.3 ± 0.05 2.2 ± 0.06 1.4 ± 0.04 ~,*** (g/dl) (1.6-2.3), 10 (1.8-2.6), 19 (2.0-2.4), 8 (1.1-1.8), 22

Tissue protein 2.5 ± 0.09 2.2 ± 0.2 1.8 ± 0.1 1.7 ± 0.07 ,L* (g/dl) (2.2-3.0), 11 (1.6-3.2), 10 (1.3-2.5), 10 (1.5-2.1), 10

Albumin 1.1 ± 0.06 0.9 ± 0.09 0.8 ± 0.04 0.6 ± 0.04 $** - (g/dl) (0.7-1.4), 10 (0.6-1.4), 10 (0.5-1.0), 11 (0.4-0.8), 12

Peritoneal fluid albumin 1.18 ± 0.08 1.18 ± 0.09 1.23 _+ 0.08 1.0 ± 0.07 (g/dl) (0.9-1.6), 9 (0.8~1.6), 8 (0.8-1.4), 7 (0.7-1.4), 11

Albumin-excluded 0.23 _ 0.02 0.24 _+ 0.03 0.33 ± 0.01 $* 0.43 ± 0.02 $*** volume fraction (0.12-0.3), 9 (0.13-0.34), 8 (0.3-0.35), 7 (0.34-i1.55), 11

Tissue wet weight 451.4 _+ 28.3 512.5 ± 31.2 645.4 ± 42.2 701 ± 54.6 $* (#g) (295-673), 12 (388-743), 10 (444-863), 10 (454-1040), 12

Tissue dry weight 76.5 _+ 5.8 88.3 ± 6.1 121.1 ± 8.9 135.2 _+ 12 1"** (#g) (50-111), 12 (59-126), 11 (92-172), 10 (82-222), 13

Hydration 5.1 ± 0.2 4.7 ± 0.2 4.2 _+ 0.1 4.1 ± 0.1 ,L** (#g water/#g dry wt) (4.2-5.8), 12 (3.9-5.4), 10 (3.6-4.6), 9 (3.2-4.8), 12

Note. The table contains values of the parameter in the format: mean _+ SEM, (range), n. ,L, decrease; ?, increase.

* P < 0.05, **P < 0.01, ***P < 0.0001, when compared to 210 day old rats.

in the per i tonea l f luid samples d id not va ry s igni f icant ly wi th age. T h e r e w e r e no

s igni f icant d i f fe rences b e t w e e n any age g roups ( A N O V A , p < 0.16) and there was

no cor re la t ion wi th age ( r = - 0 . 2 ) . R e g r e s s i o n analys is y i e lded a s lope o f - 0 . 0 0 3 _+

0.001 g/dl per 10 days bu t was not s ignif icant (P < 0.16).

Excluded Volume Fraction for A lbumin

There were s igni f icant d i f fe rences in e x c l u d e d v o l u m e b e t w e e n age groups

( K r u s k a l - W a l l i s test g a v e P < 0.0001). E x c l u d e d v o l u m e f rac t ion was cor re la ted

wi th age, r = 0.8. R e g r e s s i o n analysis y i e l d e d a s ignif icant (P < 0 .0001) pos i t ive

s lope o f 0 .004 _+ 0 .00003 per ~10 days. ' H o w e v e r , the re la t ionship m a y be non l inea r

s ince there was no s igni f icant d i f fe rence b e t w e e n 80- and 210 -day -o ld rats (us ing

e i the r mul t ip l e c o m p a r i s o n s or a s imple t test). R e g r e s s i o n analysis o f the last three

age groups ( 2 1 0 - 6 3 0 days) gave, a s ignif icant (P < 0 .0001) s lope o f 0 .0044 _+ 0 .0004

per 10 days (Fig. 1).

Chloride Concentration

Mesen t e r i c t issue shou ld be p r e d o m i n a n t l y ex t race l lu la r mat r ix ( E C M ) . H o w e v e r ,

this suppos i t ion is based on ana tomica l observa t ions . It was necessa ry to demons t r a t e

that our data re f lec ted E C M proper t ies . Fo r e x a m p l e , the wate r data cou ld not conta in

a s ignif icant ce l lu lar c o m p o n e n t s ince this w o u l d in t roduce an error into the ca lcu la t ion

o f E C M a lbumin concen t ra t ion . T h e ch lor ide concen t r a t ion o f ex t race l lu la r wa te r is

abou t 20 t imes h ighe r than that o f in t race l lu lar water . Thus , t issue ch lo r ide concen t ra -

EXCLUDED VOLUME 377

0.6

O

o 0.5 0S

0,4

0

0.3

Q 0.2 H

el "~ O.l

0.0 0

o ~ O ~ ~ I o

8 8 I ~ 111

O o 8 O

i i i i i i 100 200 300 400 500 600 7 0 0

AGE (in. days)

FIG. 1. Age-related changes in albumin-excluded volume fraction. Regression analysis yielded [(0.004 _+ 0.00003) + (0.18 _+ 0.02)] per 10 days.

tion provides a good indication of the percentage of ECM in a tissue (Lowry and Hastings, 1952).

Chloride ion concentrations in serum and mesenteric tissue samples were measured and compared to determine the ECM fraction of the mesenteric tissue. Serum chloride concentration was found to be 110.2 + 2.9 meq/liter (n = 8) while the tissue concentra- tion was 107.7 _+ 2.7 meq/liter (n = 8). The unpaired t test indicated that there was no significant statistical difference (P < 0.6) between the two groups. A nonparametric test gave P > 0.1, suggesting that there was no significant difference between the two groups.

Although there is no statistically significant difference between these two values it is worthwhile to estimate the potential range of ECM in this tissue. Clearly, no difference implies 100% ECM. The minimum percentage of ECM can be calculated by noting that intracellular chloride concentration is 5 meq/liter. Using a plasma chloride value of 107.7 meq/liter, 98% of the mesenteric tissue would be ECM. If we use the lower value of the 95% confidence interval, then mesenteric tissue chloride would be 101.5 meq/liter, in which case 92% of the mesenteric tissue would be ECM. Therefore, it appears likely that r~esenteric loose connective tissue is composed largely of extracellular matrix.

DISCUSSION

The mesenteric tissue drape is a standard anatomical preparation for the study of loose connective tissue. Loose connective tissue consists of a collagen-glycosamino- glycan matrix bounded by mesothelial cell layers. The thickness is about 17 #m; the mesothelial cells are spindle shaped and contribute little to the overall thickness (Barber et al., 1987). The intima of the vasculature, the epimysial capsule of striated muscle, the subcutis, and portions of the eye are of similar composition. The results of the chloride measurements indicate that mesenteric tissue is predominantly ECM.

The exclusion phenomenon of serum albumin from connective tissue polysaccha-

378 P A R A M E S W A R A N ET AL.

rides was first demonstrated by Ogston and Phelps (1961). Volume exclusion amplifies the effect of changes in tissue protein content on osmotic pressure thereby increasing the safety factor against edema (Granger et al., 1984). Furthermore, the reflection coefficient is proportional to the square of the excluded volume fraction. Therefore, increases in excluded volume indicate increased hinderance to protein transport.

We cannot find any data about age-related changes in albumin-excluded volume fraction. However, there are a number of in v ivo measurements to compare our data with. Barber et al. (1990) reported a value of 0.22 for albumin exclusion in rat mesentery interstitial matrix. Studies on the effect of varying superfusate albumin concentrations (Barber et al., 1993) on rat mesentery yielded a value of 0.26 in 80- day-old rats. The albumin-excluded volume fraction for dog lung under normally hydrated conditions was 38% (Parker et al., 1979). Bell et al. (1980) estimated albumin exclusions between 20 and 30% in cat skin and muscle while Bert et al. (1982) reported a significantly higher exclusion (68-78%) for excised human dermis. Much higher values have been reported for dense connective tissues: 0.9 for cartilage (Marou- das, 1970) and 0.8 for corneal stroma in the body (Maurice, 1969).

We found a progressive increase in albumin-excluded volume fraction in rat mesen- teric tissue with age (Fig. 1). The reason for this increase may be attributed to age- related changes in matrix physicochemical properties. The primary factors that may contribute to this increase are changes in hydration, matrix collagen, and glycosamino- glycans (e.g., hyaluronan).

Tissue hydration showed a significant decrease with age (see Table 1). Dehydration causes an increase in matrix density and the number of excluding domains, thus causing a decrease in the fractional distribution volume for albumin. The increase in albumin-excluded volume is about 20% for a decrease in hydration from 8 to 4 (Granger, 1981). In the present study hydration decreased from 5.1 in juvenile rats to 4.1 in old rats. This change in hydration would be expected to cause about a 5% increase in excluded volume. This effect is too small to account for the large increase in albumin-excluded volume with age (Fig. 1).

The degree of exclusion not only depends on the matrix components but also on the concentration of the protein itself. The volume excluded by both hyaluronate and collagen is higher for lower albumin concentrations (Wiederhelm et al., 1976). They reported that the volume exclusion was most pronounced at low plasma protein concen- trations and varied with the concentration of collagen. If tissue albumin concentration decreased in proportion to the decrease in serum albumin then it would have changed from 0.92 g/dl in mature rat mesentety to 0.65 g/dl in old rat mesentery. The increase in albumin excluded volume that would have been effected by this change was esti- mated from Wiederhelm et al. (1976) as 1.3%. However, rthis effect is too small to account for the 80% increase in albumin-excluded volume with age (Fig. 1).

Laurent (1964) found that the excluded volume for albumin increased with an increase in hyaluronic acid concentration. Although the glycosaminoglycans have a greater effect than collagen on plasma protein exclusion (Aukland and Nicolaysen, 1981), the contribution of collagen to this phenomenon cannot be discounted due to its larger concentrations in certain tissues such as the dermis. Significant exclusion of albumin has been reported for collagen mixtures of various concentrations (Wieder- helm et al., 1976). Katz and Li (1973) determined the exclusion of a polymer consider- ably smaller than the plasma proteins by collagen. The cross-linking hypothesis of aging may explain, in part, the observed increase in albumin exclusion. Heikkinen

EXCLUDED VOLUME 379

(1969) found that c o l l a g e n c ross - l inkages in rattail doub led b e t w e e n 21 and 700 days

o f age. This i nc reased c o l l a g e n c ross - l ink ing m a y ampl i fy the e f fec t s o f steric exc lus ion

by l ower ing the f rac t ion o f total mat r ix wa te r v o l u m e avai lable .

Ra t mesen te r i c t i ssue hydra t ion , total pro te in , and a lbumin concen t r a t ion dec rease

b e t w e e n 210 and 630 days o f age. The a l b u m i n - e x c l u d e d v o l u m e o f this loose connec -

t ive t i ssue increases o v e r this age range. Th is c h a n g e in exc luded v o l u m e is too large

to be accoun ted for by dec reases in hydra t ion and a lbumin concent ra t ion . This sugges ts

that the increase in the e x c l u d e d v o l u m e f rac t ion for a l bumin is due to age- re la ted

changes in mesen te r i c t i ssue g l y c o s a m i n o g l y c a n s or co l lagen .

A C K N O W L E D G M E N T S

This work was supported by NIH Grant R01 AG10257 and THRI. The technical assistance of Rancie Hannah and Patrick Walsh is gratefully acknowledged.

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