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Albumin metabolism in diabetes mellitus
Clinica Cl&&a Ada, 49 (1973) ((3 Elsevier Scientific Publishing
261-275
Company,
Amsterdam
~ Printed
in The Netherlands
261
CCA 5961
ALBUMIX
~‘v~ETABOLISM
IN DIABETES
MELLITUS
SUMMARY
control
Albumin metabolism was investigated in varying detail in 22 diabetic, and 20 subjects, using preparations of albumin labelled with 1311 or lz51. Evidence is presented that the metabolic properties of the labelled preparations
were comparable with those previously reported. Turnover studies indicated that albumin left the intravascular
pool at a signifi-
cantly
increased rate in diabetics. Estimations of plasma degradation rate constant in 39 subjects, and absolute degradationrate of 25 of these subjects, indicated that degradation was not increased in the diabetics, and was not responsible for the observed changes in turnover in the subjects studied. Plasma albumin pool was not altered, but extravascular were found to be enlarged in diabetic patients. Discussion of these findings lends weight to previous that the extravascular (or interstitial) pool is a summation of and that in diabetes mellitus the transfer of albumin between
pools, and total pools, reports which suggest a number of sub-pools, these sub-pools may be
interrupted.
1NTRODI:CTIOiY
Atthough albumin metabolism has been studied in great detail in normal people, and those with a variety of disease states, there are no reports of investigations into albumin metabolism in diabetic subjects. Disturbances of albumin metabolism in diabetic patients include the hypoalbuminaemia and oedema of diabetic nephropathy, transient oedema occurring in some diabetics when insulin treatment is started, or control re-established, and albumin leakage from small vessels in the eye and kidney. In diabetics with severe angiopathy, excess albumin has been detected in the saliva (P. J. Scott and H. K. I. Ibbertson, unpublished observations). Using a preparation of 1311-labelled albumin that was unsuitable for most metabolic work (chloramine-T labelling), but probably valid for early distribution studies, Ismail et al.” showed that early (first 3 11) plasma clearance was reduced in a diabetic group compared with control subjects, and postulated that this indicated decreased capillary permeability to
albuminl, although their with either increased or described a fall in serum quate in protein, but very
results may be interpreted in ways which are compatible decreased capillary permeability. foles and MacDonald” albumin concentration in healthy adult men fed diets aderich in carbohydrate, but they did not undertake any tracer
studies. Coles and her group have not published results from diabetic patients. The present studies were undertaken to determine whether there were any’ disturbances of albumin metabolism in diabetes mellitus which could be demonstrated by standard tracer techniques. In particular, the experiments were designed to test the possibility that excessive leakage of albumin might occur from the plasma pool to an extent sufhcient to produce secondary adjustments in synthesis, catabolism and plasma pool mass. The increased “capillary fragility” in the retina with the associated plasma exudation, together with the pllenornen~~ll of diabetic nepllr[)~tl~~, suggested the possibility that the normal rate of albumin catabolism within the gastrointestinal tract might be increased in diabetic subjects. This possibility was also investigated, to some extent in parallel with the studies reported in the present paper, but the results are being reported separately”. This paper, therefore, describes studies made of albumin turnover and degradation in agroup of diabetics, and in a group of non diabetic control subjects. Differences were found in albumin turnover in the diabetic patients. The rate of transfer of albumin to the extra vascular pool (or pools), was found to differ between the two groups of subjects. MATERIAL
ANI)
METHODS
Subjects .&died: These were mostly hospital in-patients, who were in a steady state in terms of plasma albumin levels, Hb, PCV, body-weight, diabetic control and general clinical measurements. Two groups were studied; the mean age for both groups was 60 years. 1Vovz diabetic control grout These were mostly convalescing after myocardial infarction. Glucose tolerance tests were not performed, because of the difficulties of interpretation at this stage of convalescence in patients, but glycosuria, or a history suggestive of diabetes, was carefully excluded. The term “normal subject” has been avoided, although several could be regarded as such.
These were a heterogeneous group of patients with overt diabetes. Many were in the older age groups, and their diabetes was of short duration, or mild, but one had been well controlled on insulin for 47 years. Two with severe nephropathy were 37 and 42 years of age, and another who had developed severe neuropathy was only 24 years of age. Preliminary inspection of results revealed no differences between the insulin and non insulin dependent groups, and results for diabetics were therefore analysetl as one set of data.
~5% Cohn Fraction
v human
serum albumin
(HSA) was iabelled with VJlI or
ALBUMIN
METABOLISM
IN DIABETES
rrsI using a modification
263
of McFarlane’s
jet iodination
iodine monochloride
method4,
to produce radio-iodinated HSA (RIHSA). Labelling efficiency varied between 35 and 70% (mean 53:/J, with substitution of iodine between 0.5 and 1.0 atoms per molecule, a level compatible with minimal denaturation of this protein. Protein binding, b! trichloracetic acid precipitation, was in excess of 97.5%. Carrier HSA was added to bring the final protein content to greater than I%, thus reducing denaturation b! auto-irradiation. All preparations were used within 18 11 of labelling. Albumins estimation Plasma albumin was estimated by a bromcresol green method, HCl buffer in place of the more usual citric acid citrates.
using a glycine
Albumin standard 25% Cohn Fraction v HSA was diluted to approximately 556 go/, and standardised as total protein using a biuret method, and a Hyland Laboratories protein standard. PROCEDURE OF IN-VIVO
STUDIES
Thyroidal uptake of iodine was blocked tion before and during the study. Heparinised
by giving 507; potassium iodide solublood was taken at 15 min after intra-
venous injection and then daily at the same time for at least seven days, although many studies were continued for ten days or more. Plasma was separated for counting, and aliquots frozen for albumin estimation. Total urine collections were made in 21-h periods
for seven Subjects
singly,
but
days.
were
the
studied
majority
Approximately
IOO ,&i
Radioactivity
was adjusted
albuminuria, 20%
radioactivity
trichloracetic
counts
of
available. control
The
subjects
first
few
and
diabetic
were
studied patients.
Ia11 was used, and 25-100 ,uCi l25I. gamma
and urine samples
spectrometer
to ensure present
a counting as intact
acid precipitation.
due to lz51 and 51Cr were
CALCULATIONS
became
in pairs
of 5 ml plasma
series 4200 single channel of counting
as they
were
Where
separated
using
a Philips
with a three inch well crystal.
was determined
Duration
accuracy RIHSA %r
of _t: 1%.
was albumin
by pulse
In the presence
determined
height
using
of
ice-cold
was used simultaneously, analysis.
AND DEFINITIONS
Turnover results were calculated on the basic assumption that the RlHSA was being distributed within and catabolised directly from, one exchangeable pool of an open, two-sided mamillary system. It was further assumed that degradation of albumin was occurring within the “intravascular (i.v.) compartment”, or in tissue that equilibrated rapidly with the plasma, that newly synthesised albumin was released into this same plasma compartment, and that the extra-vascular (e.v.) compartment functioned only as a reservoir in which albumin, either as HSA or RIHSA, was not metabolised. The mathematical analysis used was that described by Campbell et 01.6. Plasma uohme (ml) = SVD/ A15 x 1.015
where S = specific activity of standard, V = volume of labelled solution D = dilution factor of standard. Al5 =-. activity of plasma at 15 min.
Plasma radioactivity
(QPy/,) =
Activity
of plasma sample
Activity
of 15 min sample
L---
--- -- -__
given.
x 0.98 i:IOO.
Mean QpO/“:values for Qp were plotted on natural gra.ph paper against time, and the best curve fitted by eye. Mean QP for each z4 II period was taken at 0.38 day. LT:yiuavy cxcrction~ &Jo/) = percentage of radioactivity given excreted per ~4 h.
This was calculated for each 24 h period. k, was accepted as the percentage of the plasma albumin pool degraded per day, with these assumptions: (a) that the plasma radioactivity measurements derived only from isotope bound to metabolicall>~ active albumin, or to isotope released from degradated albumin, which was in the process of being excreted. (b) That urinary radioactivity measured as iodine, reflected the rate of release of isotope from enclogenous, and exogenous (gut) metabolism of RIHSA, once adequate time had elapsed for the total body iodide pool to equilibrate with labelled iodide?. Thus radioactivity measurements used in the calculations were derived from non-protein bound isotope. (c) That urinary excretion of iodide represented the sole route of isotope loss from the body, iodide released from intestinal metabolism being excreted in the urin@. These assu~~l~tions were clearly not met for the first ~4-11 period when iodide pools were e~luilibratin~, and plasma levels falling rapidly. During this period free iodide present in the preparation injected intravenously, and isotope from damaged fragments were also being excreted. The value for the first z-11 period was, therefore, omitted
when calculating
mean k,.
Mean k, was calculated as the mean value for days I-Z to 667 inclusive, except that one, or at the most two, days were omitted from calculation when urine collections were known to he incomplete. Plasma a~bu;~z~~~, f~ool ~~~~~2s) =: plasma volume (ml) x plasma albumin concentration (g%)/roo.
Equilibriunz
tinzc
For a two compartmental system, total pool size can be determined if “equilibtransfer is zero, and rium time” is known. At that time net inter-compartmental the rate of decrease of the plasma compartment is due to metabolic degradation only. exThe relationship th (clays) = 0.693/k, was used to obtain the slope representing ponential decline in RIHSA as an indicator of the rate of metabolic degradation of albumin. The values for QP were plotted on semi-logarithmic paper against time, and a line with slope t k was plotted tangentially to the curve Qp. The point of contact defined ~~lLlilibr~~ln1Time TE (days) (Fig. I).
265
ALBUMIN METABOLISM IN DIABETES
100 90
80 70 80
WX 50
40
30
Fig.
I.
Showing the method used to calculate equilibrium
time TE days. (For details see text).
Total ~Z~~~i~ pool (g) = plasma albumin pool (g)+(Q’P{-Q’E)/Q’P. Where Q’P represents the value of Qp and Q’E represents the value of QE at equilibrium time. QR, the percentage of the dose retained in the body (i.e. the result of subtracting each day’s urinary excretion from the portion of the dose left in the body the previous day), minus the percentage remaining in the plasma pool (Qp), gave, for each day, the percentage of the dose remaining in the extra vascular fluid (QE). Extra vascular albuvttin $ool = total pool minus plasma pool. Biological
half life --T$
0.693 x total albumin pool (g) (days) =: Metabolic degradation rate (g/day-‘j
Secretion rate = In the steady rate plus other loss. It is probably valid to in the presence of a marked secretion rate, and certainly minimum value for secretion
state,
secretion
rate equals plasma degradation
assume that in most subjects in the steady state (except protein-losing enteropathy), degradation rate will equal valid to assume that degradation rate will represent a rate.
RESULTS (I) Biological half life was in the range 12-35 days for II control subjects (mean 21.1 days). This is comparable with those reported by other workerss~lO.
.MCPHERSO?i
266
(2) Uriwwy radioactivity. Approximately ~-7% of the dose was excreted in the first 24 11, with a maximum of 12.3 y/o(in keeping with reported experience, excretion of isotope over the first 2.4 h was lower when 1251 had been injected). This retlccts the high initial plasma radioactivity, excretion of iodine not bound to protein and cataholism of albumin damaged in labelling. (J) Depadatiolz mtc. The mean value obtained for II control subjects was ISI mg kg-l day-l (I S.II).62.2), and for fourteen diabetics 183.5 mg kg-l day-1 (I S.D.6z.H). This was comparable with values of 185 mg kg-’ day-1 obtained by Cohen ct al.s, by Dykes**, although 202 mg kg-ldayr by Reekcn et c~l.lr and 204 mg kg-‘day-r rather higher than Takeda
and Reeve’s
valuers of 142 mg kg-%lay-mI.
Turnover studies 42 Subjects were studied, 22 diabetic and 20 control. Details are given in Table I. Fig. 2 shows the values for mean Qp for days O-I and 6-7, and it can be seen that the grouping, while identical for o-r, differs at day 67. Fig. 3 shows the mean of the values for QP for each group on each of these days. A progressive divergence between the curves was noted from day z-3 onwards. Significance (Students’ t test) increasctl from fi --= 0.05 at dav 223 to 0.001 < /, < 0.002 at day 6-7. It was concluded that Iabelled albumin was leaving the plasma at a greater rate in diabetic, than nondiabetic subjects.
(‘ontrol mean S.D. Diabetic mean SD. Significance (Student’s t) at 50/b level
60 IL.” 60 16.1
3’Tcl 781
45.03 12.16
72.1
49.5
6.24
g.16
40.3 4.65
35 3 4.46
jr.1 -1.77
29.9 3.93
I8.L 3.78
9.39 ‘?.oj
2836
32.29
71.X
47.9 4.98
37.2 4.60
31.6 4.52
2S.r 4.23
r-5.7 4.01
L3.8 3.79
IO.XS
7x0
5 ‘5.i
5.83
___ ._ ____-
yes yes YCS (details in text)
I.C).j
yes
Plusma degradations rate constant (k,). Iior the 39 subjects with satisfactory urinary collections, the mean values for k, are shown in Fig. 4 and Table I. There are no significant differences between the groups. Plasma
degradation rate A smaller number of subjects were fully studied because unsuspected difficulties occurred with plasma albumin estimation and plasma from some studies were discarded before this potential error was discovered. Results for II control, and 14 diabetic subjects were not affected, and these results are shown in Table II. Two features emerged. I%rstly, the mean degradation rates of 181.2 mg kg-‘day- 1 for controls, and 183.5 mg kg-‘day-1 for diabetics were virtually identical. For the II control subjects separately (Fig. 6) and for the 25 control plus diabetic patients con-
ALBUMIN
METABOLISM
IN DIABETES
267
CJP % 100,
90. . 80.
.?Q *:* ;
70.
:s
I* .5J.-
60.
50.
40.
30.
20.
1 1
2
3
4
5
6
7
Fig, 2. Turnover data for control and diabetic subjects showing divergence of the two groups by days 6-7.
sidered together (Fig. 7), a linear relationship was demonstrated between plasma albumin level and degradation rate (r = 0.76 for the fitted regression line in Fig. 6, and Y = 0.68 for Fig. 7). The calculated regression line intersects the abscissa for both sets of data at the serum albumin level of 3 g/roe ml. Secondly, not only did the mean values for the two groups coincide, but the range of distribution was also similar (Fig. 5). Secretion rates In Fig. 7, the values for degradation rate also equal minimum values for secretion rate, except for the two diabetic subjects with heavy proteinurea. In these two cases, the addition of urinary protein gave the values shown by the double circles. This is in accord with previous published evidence I4 that secretion rate is not related either to plasma concentration of albumin or to degradation rate. It was concluded that most diabetics had normal secretion rates, although in some secretion rate was increased. Pool distribution Details of plasma, extravascular, and total pool size are given in Table II. Extravascular pool size was 3.16 g/kg for control, and 3.54 g/kg for diabetics. This increase in size of the exchangeable extravascular albumin pool in diabetic subjects
MCPHERSON
Fig. 3. Mean values for Qp are shown for each 24 h period as the mean of the control and diabetic groups. The progressive divergence of these turnover curves can be seen, and the degree of significance, using Student’s t test is shown where appropriate.
was significant when compared with that of the control subjects (0.01 < p < 0.02). The increase in the extravascular pool size was reflected in the total pool size, but the differences between total body albumin pools were not statistically significant. Diabetic (1.89 g/kg) and control (1.83 g/kg) plasma pool sizes were not significant11 different. Plasma albumin concentration No significant difference was observed, mean values being 4.85 mg% 0.52 g%) for controls, and 4.63 g% (I SD. 1.03 g%) for diabetics.
(I SD.
Plasma vohmes Mean values obtained for the small series were 37.63 ml/kg for control, and 41.25 ml/kg for diabetics, and .this difference was not significant. The values for the whole series are given in Table I, but the difference between the means of 45.03 ml/kg (control), and 39.29 ml/kg (diabetic) was again not significant at the 59/o level (0.05 < $ < 0.10). Diabetic status Four of the patients
studied had been clinically
diabetic
for 15 years or more.
ALBUMIN METABOLISM Ipi DIABETES
269
There appeared to be no relationship between length of diabetes and the parameters studied. This was not entirely unexpected in view of the known lack of correlation between length of diabetes and extent of complications. An attempt was made to relate QP and k, to presence, or absence of retinopathy, peripheral neuritis and nephropathy. No clear-cut relationship emerged. There appeared to be no relationship to method of treatment. ALBUMIN MEAN
VALUE
PLI \SMA
RATE
RATE
CONSTANT
400
IKll
18 17
DEGRATION
GIAGETES
CONTROL
I
DIABETES
16 P E A
.
15
300
14 13
C E N T
P E R
.
12
.
11
.
10
M G
. .
/
.: :. .
9 8
/
l
7 0 A Y
.
l
K G
200
0 A Y
: . .
.
.
5
.
.
.
6
:
100
4
.
3 2 1 Fig. 4. The mean value for plasma rate constant difference between the groups was demonstrated. Fig. 5, Results for calculated tween groups. TABLE FULL
degradation
for each of 39 studies is shown. No significant
rates are shown. There is no apparent difference be-
II
STUDIES
Control (n =
Plasma Pools in g/kg albumin Plasma Extravac. concentralion (g%)
57.5 12.2
4.85 0.52
1.830 0.354
3.161 0.608
4.992 0.705
181.2 62.2
57.5 14.6
4.69 1.03
1.889 0.469
3.542 I.000
5.460 I.329
184.1 62.8
No
No
No
Yes No (P<0.02>0.01)
Total
i.V./fXl.
ratio
II)
S.D. Diabetic (n = S.D. Significance (Student’s
Degradation rate (mglkglday)
Age (Y)
I
: 1.791 0.496
14)
t)
No
I :
1.886 0.414
No
MCPHERSON
270
PLASMA
ALBUMIN
CONCETRATIGN
(G./
100
ML.1
Fig. 6. Values for calculated degradation rate are plotted for each of II control studies, against plasma albumin concentration. A line of regression has been plotted.
DISCUSSION
In terms of the biological half lives, urinary isotope excretion data, and calculated degradation rates detailed above the RIHSA used in these studies met the criteria established by other workers for satisfactory radio-iodinated protein. The study was designed to provide comparative data. For the large majority of studies, control and diabetic subjects were paired, each receiving an aliquot of the same freshly prepared RIHSA. This not only helped provide a check that an individual labelling was satisfactory, but also validated comparisons between results for the two groups. Although it is known that the HSA standard in fact contained up to 4% of peptide content as globulin, the value for the total protein was taken as that for albumin, which is more satisfactory than might be thought, as difficulties in obtaining adequate albumin standards are not always recognisedlO. Differences in dye binding occur between different speciesl5, and Peters” recommends use of human mercaptalbumin of Fraction v as standards, if allowance is made for about 4% impurity in the latter. Degradation of albumin There was no significant difference between groups for degradation rate, and this is in keeping with the results for degradation rate constant (k,) for the larger
ALBUhIlN
METABOLISM
DEGRADATION 400
IN
271
DIABETES
RATE
I
_
300,
j 200, z A Y
100.
1.96s - 65.16 0
1
PLASMI
I
2 A~E,“M,N
I 3
I 4
CONCENTRATION
5
6 IG/
100
ML I
Fig. 7. Shows the values for control l and diabetic o studies plotted together. The dotted lines represent * 1.96 S.D. These values for degradation rate also equal secretion rate, when there is known to be no faecal or urinary loss. This is the case for all subjects except the two with heavy proteinurea. The value for secretion rate in these two is shown by a double circle 0.
series of 39 subjects. It was interesting to note that the relationship between plasma albumin concentration and degradation rate was of the same order as that shown by Andersen and Rossing16 (data plotted by Peters”). The line of regression does not go through the origin, suggesting that some other factor, or factors, are involved, over and above a direct increase in degradation rate, with increase in plasma albumin concentration, e.g. a superimposed additional catabolic rate increasing exponentially above 3 g/roe ml. No relationship was found between plasma albumin concentration and total, or e.v., albumin pool mass, or between degradation rate, and total, or e.v., pool mass. Turnover studies It is somewhat suprising that any difference at all should have emerged when the nature of the diabetic group is considered and even more that the differences should be so clear-cut. The diabetics were a very mixed collection of patients, ranging from those with newly discovered diabetes, without obvious complications, to diabetics of so-40 years standing, some with, and some without complications. Nevertheless, the turnover studies revealed one clear cut point of difference between the groups. The rate of clearance of label from the plasma was increased in the diabetics from day 2-3 onwards, the difference became greater as time passed, and it is reasonable to
272
MCPHERSON
accept that this represented clearance of labelled albumin. Albumin could leave the intravascular compartment at a greater rate by a number of different mechanisms. (a) Increased degradation: This was not demonstrated. In the 39 studies with adequate urine collections, the plasma degradation rate constant (k,), showed no difference between groups. In the smaller number in which absolute degradation rates were measured, there was also no difference. (b) Increased loss into another pool, or pools. The demonstrated overall increase in extravascular pool size reflected the tcndencv to increased total body albumin pool size in many, but not all, diabetics. Those with the largest pool sizes tended to have vascular complications, but the converse was not exclusively true. It would be reasonable to explain the turnover data by this difference, with increased rate of transfer into a larger pool, but for the observed fact that the difference between the groups continued to increase beyond equilibrium time. Equilibrium time itself was the same for the two groups. This suggested either an alternative route of loss from the plasma compartment, or that the two compartment system provided an invalid model for analysis of the turnover data. Other route of loss from i.v. comfiartvaents The model described envisaged only one route of loss from the total system. Loss in one direction into any further compartment was not compatible with this model. The most likely abnormal routes for loss of albumin from the intravascular compartment are urine, faeces or skin. Urine: Significant urinary protein loss was detected in only two patients. Removal of these two patient’s turnover data does not affect the validity of the between group differences, the mean value of QP at day 6-7 now being 24.45%, and 0.002 < $ < 0.01. Calculations showed that occasional minor incidents of proteinuria had not contributed significantly to the differences noted between control and diabetics. Faecal loss of jvotein: Isotope released from RIHSA secreted into the gastrointestinal tract is re-absorbed and excreted in the urines. If the faecal loss of albumin was significantly increased it would be expected that a significant increase in urinary radioactivity would occur, and that this would be reflected by increased values for fractional, and absolute degradation rates. This did not occur, and it is therefore most unlikely that the differences in plasma turnover are due to increased faecal loss. and the results suggested that there is Further work was undertaken using Xr-HSA, little or no increase in gastrointestinal protein loss in diabetic patients”. Skin: Loss of albumin by way of shed skin, or sweat, would not be reflected in degradation rate, and the label from the albumin would not be collected. However, prolonged in viva studies, using radioiodinated proteins, have shown that 95% or more of the isotope originally injected is recovered in the urine17, providing indirect evidence that loss from the skin is not excessive. Alternatively, there could be alterations in rate of entry and exit of albumin to the skin compartment (or subcompartment), as discussed below. Alternative model fey albumin distribution : To date calculations have been based on the assumption that the extravascular compartment was, or behaved as though it was, homogenous, although it has been known to consist of a large number of tissues and serous fluids. Rothschild et aZ.l8 showed that skin, muscle and viscera account for
ALBUMIN
over 60%
METABOLISM
IN
of extravascular
DIABETES
albumin,
273 but the assumption
that
these behave
as one
compartment is open to doubt, and reference to Fig. I of their paper suggests that there may be different equilibrium times for skin and muscle. Doubt has been thrown on this simple model by a number of workers, and a large variety of complex mathematical models have been considered and adopted, or rejected, on the basis of experimental evidence4~7~13~19.Takeda and Reeve13, and Reeve and BaileyI@, described mathematical methods for determining exchange rates between various compartments, and subcompartments, by which they were able to determine values for flow rates through the extravascular compartment. They proposed a similar overall model to that used in this paper, but albumin molecules were regarded as flowing in innumerable discrete streams through the interstitial fluids, with different rates of movement. An interesting, but unexplained, observation in Takeda and Reeve’s paper, was that, quite commonly, a small bump on the turnover curve was noted between day 4 and 8. This also occurred frequently in the present studies, and although the explanation is not obvious, it raises the possibility of reentry to the vascular pool from a pool of large mass, but slower turnover time. Reference to Fig. z of the paper by Katz et uLZo, raises the possibility that this could be carcass (which is mainly muscle). Katz et al. have published two papers which cast doubt on the value of all plasma protein tracer work published to date, and they challenge the conclusions reached by previous groupPyZ1. The validity of the conventional multi-compartmental model of albumin metabolism is questioned, and in particular they calculate that in both rat and human, RIHSA studies lead to underestimation of e.v. mass by 20--40~/~. They studied rats*O, and humansZ1. Extracts of tissue were made and albumin measured by a number of methods. In rats simultaneous RIRSA (radioiodinated rat serum albumin) studies were made in some experiments, and RIRSA was used to determine occluded blood in tissues. Disagreement with their methods could include doubts over the metabolic properties of their labelled albumin, their lack of allowance for sequestration and perivascular circulation of albumin22, and difficulty in knowing the state of the albumin they measured. The methods used were electrophoretic or immunological, and no assessment was made as to the metabolic behaviour of the substance measured. It is possible that some of the protein measured as albumin was, in fact, different from circulating albumin. It could have been of different, although similar, molecular structure, or might have been in a bound form, possibly to cell membranes or other proteins. Albumin may be found inside cells, and this would be included in Katz’ tissue extracts. Such “albumin” might be unavailable for, at least, short term turnover, and be additional to the exchangeable albumin. In concluding their second pape+, in which they reported no isotope studies, Katz et al. concluded that “total extravascular albumin and the distribution among pools determined from the plasma curve and the calculated exchange rates, have little or no physiological meaning”. The alternative interpretation is that in fact the isotope studies do indicate the physiological state, at least in the short and medium terms covered by most experimental work, and that conclusions can be reached regarding the exchangeable pools. It is possible that in the long term there is an exchange with the remaining static pool demonstrated by these workers, although the alternative is that the measured discrepancy indicates non-exchangeable, bound or functionally different albumin, or albumin like molecules. These criticisms and reservations do not alter the relevance of some of their
274
R~~PHERsoE~
findings. They demonstrated clearly that there are differences in the albumin content of different tissues, and in the exchange kinetics between circulating albumin and the extracellular fluid of muscle, skin and gut. This is in keeping with studies in rats23 and mice”* in which equilibrium was shown to occur at varying rates between plasma and different tissues, (although these papers do not agree in all respects) and also in humans18. It thus seems very likely that the described model used in the present and other papers is unsatisfactory. A reasonable conclusion that may be drawn from the above discussion is that the e.v. compartment comprises a number of sub-compartments that behave overall as one pool, if study is not too detailed, or disease states do not disturb transfer kinetics. Diabetes mellitus is a disease, or syndrome, in which disturbance of transfer across cell membranes and vascular walls is known to occur for small molecules such as glucose. It is possible that Ismail et al.’ may have observed a change of permeability at one intercompartmental boundary that may also occur elsewhere. Such a decreased rate of passage from the iv. space does not explain the excessive rate of disappearance in the longer term studies of the present series, but interference with transfer into other sub-compartments, and re-entry, may lead to the effects observed in these studies. The findings of Coles and MacDonald” do not appear relevant. A short paper by ColeP reports that a fall in serum albumin concentration could be produced by very high carbohydrate diets in a further group of six men, but that this could not be reproduced in six young women. As a greater intra-vascular mass of albumin, and greater rate of synthesis has been shown in normal men, but no difference in catabolic rate or distribution between the sexes, it is possible that Coles’ work reflects changes in synthesis. In the present studies no alteration of degradation rate was observed, and the only two patients with increased synthetic rates had considerable proteinuria. High carbohydrate diets may well alter serum albumin concentration by decreasing synthesis in normal subjects, but Coles’ findings cannot explain the metabolic changes in diabetes
mellitus.
CONCLVSIOX
If the concept of a homogenous extravascular pool is valid, or an acceptable approximation to reality in normal subjects, it is not applicable to diabetic patients. In view of the decreased plasma albumin pool and plasma concentration that can occur in patients with the nephrotic syndrome in association with an actual increase in total body albumin, intercompartmental transfer warrants further investigations. This is important in terms of therapeutics, particularly for patients with the nephrotic syndrome. If, in fact, the work described by Katz et a1.20921demonstrates the presence of a large subpool of inaccessible, or slowly exchangeable, albumin, it would be of great interest to know its function and the factors controlling its accessibility. Further study in diabetics could be of assistance in determining the primary patlmiogy at capillary and tissue level. ACKNOWLEDGEMENTS
I wish to thank the subjects
who made this study possible.
They gave their full
ALBUMIN
METABOLISM
informed
consent,
IN DIABETES
and suffered
a certain
275 amount
of discomfort
and inconvenience.
I also wish to thank my colleagues who consented to the study of patients under their care. The Hawke’s Bay Medical Research Foundation, The Royal Australasian College of Physicians, and the Bank of New Zealand made grants in support of this work, and the Hawke’s Bay Hospital Board provided laboratory space. In particular, I wish to thank Professor P. J. Scott, Auckland, for his invaluable advice and criticism. REFERENCES A. A. ISMAIL, K. KHALIFA AND K. R. MADWAR. Lancet, ii (1965) I - “I 810. B. L. COLES AND I. MACDONALD, Clin. Sci., 30 (1966) 37. 1. G. MCPHERSON, Clin. Chim. Acta, 49 (1973) 225. A. S. MCFARLAXE, Biochcm. J., 62 (1956) 135. I. G. MCPHERSON AND D. W. EVERARD, Clin. Chim. Acta. 17 (1972) 112. R. M. CAMPBELL, D. P. CUTHBERTSOX, C. M. MATHEWS AND’A. S. MCFARLANE,
Int. J. Appl.
Radiat., 1 (1965) 66. X. S. MCFARLANE AND A. KOJ, J. Clin. Invest., 49 (1970) 1903. t: E. MILHAUD, in H. PEETERS (Ed.), Protides of the Biological Fluids, 9 IO II
I*
13 14 15 16 17 18 19 20 2.1 22
Elsevier, Amsterdam, 1964, p. 280. 5. COHEN, T. FREEMAN AND A. S. MCFARLANE, C&in. Sci., 20 (1961) 161. T. PETERS, JR., Adv. Clin. Chem., 13 (1970) 37. W. L. BEEKEN, W. VOLWILER, P. D. GOLDSWORTHY, L. E. GARBY, W. E. REYNOLDS, R. STOGSDILL AND R. S. STEMLER, J. Clin. Invest., 41 (1962) 1312. P. W. DYKES, C&z. Sci., 34 (1968) 161. Y. T.+KEDA AND E. B. REEVE, J. Lab. Clin. Med., 61 (1963) 183. M. A. ROTHSCHILD, M. ORATZ AND S. S. SCHREIBER, Amev. J. Dig. Dis., ‘4 (1969) 711. F. L. RODKEY, Arch. Biochem. Biofihys., 108 (1964) 510. S. B. ANDERSEN AND N. ROSSING, Stand. J. Clin. Lab. Invest., 20 (1967) 183. P. J. SCOTT, Serum Beta-lipoproteins in Health and Disease, Thesis, University of Birmingham, 1962. M. A. ROTHSCHILD, A. BAUMANN, R. S. YALOW AND S. A. BERSON, J. Clin. Invest., 34 (1955)
1354. B. B. REEVE AND H. R. BAILEY, J. Lab. Clin. Med., 60 (1962) 925.
J. Katz, G. BONORRIS. S. GOLDEN AND A. L. SELLERS, Clin. Sci., 39 (1970) 705. J. KATZ, G. BONORRIS AID A. L. SELLERS, Clin. Sci., 39 (1970) 725. C. H. BAKER, Amer. J. Physiol., 216 (1969) 368. 23 W. C. DEWEY, Amer. J. Physiol., 197 (1959) 423. 24 A. SASSEN. A. M. REUTER, F. KENNES, M. TELIA AND C. DELCROIX, Rev. Euro+. BiOl., XVI (1971) 912. 25 B. L. COLES, Proc. Nutr. Sot., 28 (1969) 63A.
Etud. Clin.
261-275
Company,
Amsterdam
~ Printed
in The Netherlands
261
CCA 5961
ALBUMIX
~‘v~ETABOLISM
IN DIABETES
MELLITUS
SUMMARY
control
Albumin metabolism was investigated in varying detail in 22 diabetic, and 20 subjects, using preparations of albumin labelled with 1311 or lz51. Evidence is presented that the metabolic properties of the labelled preparations
were comparable with those previously reported. Turnover studies indicated that albumin left the intravascular
pool at a signifi-
cantly
increased rate in diabetics. Estimations of plasma degradation rate constant in 39 subjects, and absolute degradationrate of 25 of these subjects, indicated that degradation was not increased in the diabetics, and was not responsible for the observed changes in turnover in the subjects studied. Plasma albumin pool was not altered, but extravascular were found to be enlarged in diabetic patients. Discussion of these findings lends weight to previous that the extravascular (or interstitial) pool is a summation of and that in diabetes mellitus the transfer of albumin between
pools, and total pools, reports which suggest a number of sub-pools, these sub-pools may be
interrupted.
1NTRODI:CTIOiY
Atthough albumin metabolism has been studied in great detail in normal people, and those with a variety of disease states, there are no reports of investigations into albumin metabolism in diabetic subjects. Disturbances of albumin metabolism in diabetic patients include the hypoalbuminaemia and oedema of diabetic nephropathy, transient oedema occurring in some diabetics when insulin treatment is started, or control re-established, and albumin leakage from small vessels in the eye and kidney. In diabetics with severe angiopathy, excess albumin has been detected in the saliva (P. J. Scott and H. K. I. Ibbertson, unpublished observations). Using a preparation of 1311-labelled albumin that was unsuitable for most metabolic work (chloramine-T labelling), but probably valid for early distribution studies, Ismail et al.” showed that early (first 3 11) plasma clearance was reduced in a diabetic group compared with control subjects, and postulated that this indicated decreased capillary permeability to
albuminl, although their with either increased or described a fall in serum quate in protein, but very
results may be interpreted in ways which are compatible decreased capillary permeability. foles and MacDonald” albumin concentration in healthy adult men fed diets aderich in carbohydrate, but they did not undertake any tracer
studies. Coles and her group have not published results from diabetic patients. The present studies were undertaken to determine whether there were any’ disturbances of albumin metabolism in diabetes mellitus which could be demonstrated by standard tracer techniques. In particular, the experiments were designed to test the possibility that excessive leakage of albumin might occur from the plasma pool to an extent sufhcient to produce secondary adjustments in synthesis, catabolism and plasma pool mass. The increased “capillary fragility” in the retina with the associated plasma exudation, together with the pllenornen~~ll of diabetic nepllr[)~tl~~, suggested the possibility that the normal rate of albumin catabolism within the gastrointestinal tract might be increased in diabetic subjects. This possibility was also investigated, to some extent in parallel with the studies reported in the present paper, but the results are being reported separately”. This paper, therefore, describes studies made of albumin turnover and degradation in agroup of diabetics, and in a group of non diabetic control subjects. Differences were found in albumin turnover in the diabetic patients. The rate of transfer of albumin to the extra vascular pool (or pools), was found to differ between the two groups of subjects. MATERIAL
ANI)
METHODS
Subjects .&died: These were mostly hospital in-patients, who were in a steady state in terms of plasma albumin levels, Hb, PCV, body-weight, diabetic control and general clinical measurements. Two groups were studied; the mean age for both groups was 60 years. 1Vovz diabetic control grout These were mostly convalescing after myocardial infarction. Glucose tolerance tests were not performed, because of the difficulties of interpretation at this stage of convalescence in patients, but glycosuria, or a history suggestive of diabetes, was carefully excluded. The term “normal subject” has been avoided, although several could be regarded as such.
These were a heterogeneous group of patients with overt diabetes. Many were in the older age groups, and their diabetes was of short duration, or mild, but one had been well controlled on insulin for 47 years. Two with severe nephropathy were 37 and 42 years of age, and another who had developed severe neuropathy was only 24 years of age. Preliminary inspection of results revealed no differences between the insulin and non insulin dependent groups, and results for diabetics were therefore analysetl as one set of data.
~5% Cohn Fraction
v human
serum albumin
(HSA) was iabelled with VJlI or
ALBUMIN
METABOLISM
IN DIABETES
rrsI using a modification
263
of McFarlane’s
jet iodination
iodine monochloride
method4,
to produce radio-iodinated HSA (RIHSA). Labelling efficiency varied between 35 and 70% (mean 53:/J, with substitution of iodine between 0.5 and 1.0 atoms per molecule, a level compatible with minimal denaturation of this protein. Protein binding, b! trichloracetic acid precipitation, was in excess of 97.5%. Carrier HSA was added to bring the final protein content to greater than I%, thus reducing denaturation b! auto-irradiation. All preparations were used within 18 11 of labelling. Albumins estimation Plasma albumin was estimated by a bromcresol green method, HCl buffer in place of the more usual citric acid citrates.
using a glycine
Albumin standard 25% Cohn Fraction v HSA was diluted to approximately 556 go/, and standardised as total protein using a biuret method, and a Hyland Laboratories protein standard. PROCEDURE OF IN-VIVO
STUDIES
Thyroidal uptake of iodine was blocked tion before and during the study. Heparinised
by giving 507; potassium iodide solublood was taken at 15 min after intra-
venous injection and then daily at the same time for at least seven days, although many studies were continued for ten days or more. Plasma was separated for counting, and aliquots frozen for albumin estimation. Total urine collections were made in 21-h periods
for seven Subjects
singly,
but
days.
were
the
studied
majority
Approximately
IOO ,&i
Radioactivity
was adjusted
albuminuria, 20%
radioactivity
trichloracetic
counts
of
available. control
The
subjects
first
few
and
diabetic
were
studied patients.
Ia11 was used, and 25-100 ,uCi l25I. gamma
and urine samples
spectrometer
to ensure present
a counting as intact
acid precipitation.
due to lz51 and 51Cr were
CALCULATIONS
became
in pairs
of 5 ml plasma
series 4200 single channel of counting
as they
were
Where
separated
using
a Philips
with a three inch well crystal.
was determined
Duration
accuracy RIHSA %r
of _t: 1%.
was albumin
by pulse
In the presence
determined
height
using
of
ice-cold
was used simultaneously, analysis.
AND DEFINITIONS
Turnover results were calculated on the basic assumption that the RlHSA was being distributed within and catabolised directly from, one exchangeable pool of an open, two-sided mamillary system. It was further assumed that degradation of albumin was occurring within the “intravascular (i.v.) compartment”, or in tissue that equilibrated rapidly with the plasma, that newly synthesised albumin was released into this same plasma compartment, and that the extra-vascular (e.v.) compartment functioned only as a reservoir in which albumin, either as HSA or RIHSA, was not metabolised. The mathematical analysis used was that described by Campbell et 01.6. Plasma uohme (ml) = SVD/ A15 x 1.015
where S = specific activity of standard, V = volume of labelled solution D = dilution factor of standard. Al5 =-. activity of plasma at 15 min.
Plasma radioactivity
(QPy/,) =
Activity
of plasma sample
Activity
of 15 min sample
L---
--- -- -__
given.
x 0.98 i:IOO.
Mean QpO/“:values for Qp were plotted on natural gra.ph paper against time, and the best curve fitted by eye. Mean QP for each z4 II period was taken at 0.38 day. LT:yiuavy cxcrction~ &Jo/) = percentage of radioactivity given excreted per ~4 h.
This was calculated for each 24 h period. k, was accepted as the percentage of the plasma albumin pool degraded per day, with these assumptions: (a) that the plasma radioactivity measurements derived only from isotope bound to metabolicall>~ active albumin, or to isotope released from degradated albumin, which was in the process of being excreted. (b) That urinary radioactivity measured as iodine, reflected the rate of release of isotope from enclogenous, and exogenous (gut) metabolism of RIHSA, once adequate time had elapsed for the total body iodide pool to equilibrate with labelled iodide?. Thus radioactivity measurements used in the calculations were derived from non-protein bound isotope. (c) That urinary excretion of iodide represented the sole route of isotope loss from the body, iodide released from intestinal metabolism being excreted in the urin@. These assu~~l~tions were clearly not met for the first ~4-11 period when iodide pools were e~luilibratin~, and plasma levels falling rapidly. During this period free iodide present in the preparation injected intravenously, and isotope from damaged fragments were also being excreted. The value for the first z-11 period was, therefore, omitted
when calculating
mean k,.
Mean k, was calculated as the mean value for days I-Z to 667 inclusive, except that one, or at the most two, days were omitted from calculation when urine collections were known to he incomplete. Plasma a~bu;~z~~~, f~ool ~~~~~2s) =: plasma volume (ml) x plasma albumin concentration (g%)/roo.
Equilibriunz
tinzc
For a two compartmental system, total pool size can be determined if “equilibtransfer is zero, and rium time” is known. At that time net inter-compartmental the rate of decrease of the plasma compartment is due to metabolic degradation only. exThe relationship th (clays) = 0.693/k, was used to obtain the slope representing ponential decline in RIHSA as an indicator of the rate of metabolic degradation of albumin. The values for QP were plotted on semi-logarithmic paper against time, and a line with slope t k was plotted tangentially to the curve Qp. The point of contact defined ~~lLlilibr~~ln1Time TE (days) (Fig. I).
265
ALBUMIN METABOLISM IN DIABETES
100 90
80 70 80
WX 50
40
30
Fig.
I.
Showing the method used to calculate equilibrium
time TE days. (For details see text).
Total ~Z~~~i~ pool (g) = plasma albumin pool (g)+(Q’P{-Q’E)/Q’P. Where Q’P represents the value of Qp and Q’E represents the value of QE at equilibrium time. QR, the percentage of the dose retained in the body (i.e. the result of subtracting each day’s urinary excretion from the portion of the dose left in the body the previous day), minus the percentage remaining in the plasma pool (Qp), gave, for each day, the percentage of the dose remaining in the extra vascular fluid (QE). Extra vascular albuvttin $ool = total pool minus plasma pool. Biological
half life --T$
0.693 x total albumin pool (g) (days) =: Metabolic degradation rate (g/day-‘j
Secretion rate = In the steady rate plus other loss. It is probably valid to in the presence of a marked secretion rate, and certainly minimum value for secretion
state,
secretion
rate equals plasma degradation
assume that in most subjects in the steady state (except protein-losing enteropathy), degradation rate will equal valid to assume that degradation rate will represent a rate.
RESULTS (I) Biological half life was in the range 12-35 days for II control subjects (mean 21.1 days). This is comparable with those reported by other workerss~lO.
.MCPHERSO?i
266
(2) Uriwwy radioactivity. Approximately ~-7% of the dose was excreted in the first 24 11, with a maximum of 12.3 y/o(in keeping with reported experience, excretion of isotope over the first 2.4 h was lower when 1251 had been injected). This retlccts the high initial plasma radioactivity, excretion of iodine not bound to protein and cataholism of albumin damaged in labelling. (J) Depadatiolz mtc. The mean value obtained for II control subjects was ISI mg kg-l day-l (I S.II).62.2), and for fourteen diabetics 183.5 mg kg-l day-1 (I S.D.6z.H). This was comparable with values of 185 mg kg-’ day-1 obtained by Cohen ct al.s, by Dykes**, although 202 mg kg-ldayr by Reekcn et c~l.lr and 204 mg kg-‘day-r rather higher than Takeda
and Reeve’s
valuers of 142 mg kg-%lay-mI.
Turnover studies 42 Subjects were studied, 22 diabetic and 20 control. Details are given in Table I. Fig. 2 shows the values for mean Qp for days O-I and 6-7, and it can be seen that the grouping, while identical for o-r, differs at day 67. Fig. 3 shows the mean of the values for QP for each group on each of these days. A progressive divergence between the curves was noted from day z-3 onwards. Significance (Students’ t test) increasctl from fi --= 0.05 at dav 223 to 0.001 < /, < 0.002 at day 6-7. It was concluded that Iabelled albumin was leaving the plasma at a greater rate in diabetic, than nondiabetic subjects.
(‘ontrol mean S.D. Diabetic mean SD. Significance (Student’s t) at 50/b level
60 IL.” 60 16.1
3’Tcl 781
45.03 12.16
72.1
49.5
6.24
g.16
40.3 4.65
35 3 4.46
jr.1 -1.77
29.9 3.93
I8.L 3.78
9.39 ‘?.oj
2836
32.29
71.X
47.9 4.98
37.2 4.60
31.6 4.52
2S.r 4.23
r-5.7 4.01
L3.8 3.79
IO.XS
7x0
5 ‘5.i
5.83
___ ._ ____-
yes yes YCS (details in text)
I.C).j
yes
Plusma degradations rate constant (k,). Iior the 39 subjects with satisfactory urinary collections, the mean values for k, are shown in Fig. 4 and Table I. There are no significant differences between the groups. Plasma
degradation rate A smaller number of subjects were fully studied because unsuspected difficulties occurred with plasma albumin estimation and plasma from some studies were discarded before this potential error was discovered. Results for II control, and 14 diabetic subjects were not affected, and these results are shown in Table II. Two features emerged. I%rstly, the mean degradation rates of 181.2 mg kg-‘day- 1 for controls, and 183.5 mg kg-‘day-1 for diabetics were virtually identical. For the II control subjects separately (Fig. 6) and for the 25 control plus diabetic patients con-
ALBUMIN
METABOLISM
IN DIABETES
267
CJP % 100,
90. . 80.
.?Q *:* ;
70.
:s
I* .5J.-
60.
50.
40.
30.
20.
1 1
2
3
4
5
6
7
Fig, 2. Turnover data for control and diabetic subjects showing divergence of the two groups by days 6-7.
sidered together (Fig. 7), a linear relationship was demonstrated between plasma albumin level and degradation rate (r = 0.76 for the fitted regression line in Fig. 6, and Y = 0.68 for Fig. 7). The calculated regression line intersects the abscissa for both sets of data at the serum albumin level of 3 g/roe ml. Secondly, not only did the mean values for the two groups coincide, but the range of distribution was also similar (Fig. 5). Secretion rates In Fig. 7, the values for degradation rate also equal minimum values for secretion rate, except for the two diabetic subjects with heavy proteinurea. In these two cases, the addition of urinary protein gave the values shown by the double circles. This is in accord with previous published evidence I4 that secretion rate is not related either to plasma concentration of albumin or to degradation rate. It was concluded that most diabetics had normal secretion rates, although in some secretion rate was increased. Pool distribution Details of plasma, extravascular, and total pool size are given in Table II. Extravascular pool size was 3.16 g/kg for control, and 3.54 g/kg for diabetics. This increase in size of the exchangeable extravascular albumin pool in diabetic subjects
MCPHERSON
Fig. 3. Mean values for Qp are shown for each 24 h period as the mean of the control and diabetic groups. The progressive divergence of these turnover curves can be seen, and the degree of significance, using Student’s t test is shown where appropriate.
was significant when compared with that of the control subjects (0.01 < p < 0.02). The increase in the extravascular pool size was reflected in the total pool size, but the differences between total body albumin pools were not statistically significant. Diabetic (1.89 g/kg) and control (1.83 g/kg) plasma pool sizes were not significant11 different. Plasma albumin concentration No significant difference was observed, mean values being 4.85 mg% 0.52 g%) for controls, and 4.63 g% (I SD. 1.03 g%) for diabetics.
(I SD.
Plasma vohmes Mean values obtained for the small series were 37.63 ml/kg for control, and 41.25 ml/kg for diabetics, and .this difference was not significant. The values for the whole series are given in Table I, but the difference between the means of 45.03 ml/kg (control), and 39.29 ml/kg (diabetic) was again not significant at the 59/o level (0.05 < $ < 0.10). Diabetic status Four of the patients
studied had been clinically
diabetic
for 15 years or more.
ALBUMIN METABOLISM Ipi DIABETES
269
There appeared to be no relationship between length of diabetes and the parameters studied. This was not entirely unexpected in view of the known lack of correlation between length of diabetes and extent of complications. An attempt was made to relate QP and k, to presence, or absence of retinopathy, peripheral neuritis and nephropathy. No clear-cut relationship emerged. There appeared to be no relationship to method of treatment. ALBUMIN MEAN
VALUE
PLI \SMA
RATE
RATE
CONSTANT
400
IKll
18 17
DEGRATION
GIAGETES
CONTROL
I
DIABETES
16 P E A
.
15
300
14 13
C E N T
P E R
.
12
.
11
.
10
M G
. .
/
.: :. .
9 8
/
l
7 0 A Y
.
l
K G
200
0 A Y
: . .
.
.
5
.
.
.
6
:
100
4
.
3 2 1 Fig. 4. The mean value for plasma rate constant difference between the groups was demonstrated. Fig. 5, Results for calculated tween groups. TABLE FULL
degradation
for each of 39 studies is shown. No significant
rates are shown. There is no apparent difference be-
II
STUDIES
Control (n =
Plasma Pools in g/kg albumin Plasma Extravac. concentralion (g%)
57.5 12.2
4.85 0.52
1.830 0.354
3.161 0.608
4.992 0.705
181.2 62.2
57.5 14.6
4.69 1.03
1.889 0.469
3.542 I.000
5.460 I.329
184.1 62.8
No
No
No
Yes No (P<0.02>0.01)
Total
i.V./fXl.
ratio
II)
S.D. Diabetic (n = S.D. Significance (Student’s
Degradation rate (mglkglday)
Age (Y)
I
: 1.791 0.496
14)
t)
No
I :
1.886 0.414
No
MCPHERSON
270
PLASMA
ALBUMIN
CONCETRATIGN
(G./
100
ML.1
Fig. 6. Values for calculated degradation rate are plotted for each of II control studies, against plasma albumin concentration. A line of regression has been plotted.
DISCUSSION
In terms of the biological half lives, urinary isotope excretion data, and calculated degradation rates detailed above the RIHSA used in these studies met the criteria established by other workers for satisfactory radio-iodinated protein. The study was designed to provide comparative data. For the large majority of studies, control and diabetic subjects were paired, each receiving an aliquot of the same freshly prepared RIHSA. This not only helped provide a check that an individual labelling was satisfactory, but also validated comparisons between results for the two groups. Although it is known that the HSA standard in fact contained up to 4% of peptide content as globulin, the value for the total protein was taken as that for albumin, which is more satisfactory than might be thought, as difficulties in obtaining adequate albumin standards are not always recognisedlO. Differences in dye binding occur between different speciesl5, and Peters” recommends use of human mercaptalbumin of Fraction v as standards, if allowance is made for about 4% impurity in the latter. Degradation of albumin There was no significant difference between groups for degradation rate, and this is in keeping with the results for degradation rate constant (k,) for the larger
ALBUhIlN
METABOLISM
DEGRADATION 400
IN
271
DIABETES
RATE
I
_
300,
j 200, z A Y
100.
1.96s - 65.16 0
1
PLASMI
I
2 A~E,“M,N
I 3
I 4
CONCENTRATION
5
6 IG/
100
ML I
Fig. 7. Shows the values for control l and diabetic o studies plotted together. The dotted lines represent * 1.96 S.D. These values for degradation rate also equal secretion rate, when there is known to be no faecal or urinary loss. This is the case for all subjects except the two with heavy proteinurea. The value for secretion rate in these two is shown by a double circle 0.
series of 39 subjects. It was interesting to note that the relationship between plasma albumin concentration and degradation rate was of the same order as that shown by Andersen and Rossing16 (data plotted by Peters”). The line of regression does not go through the origin, suggesting that some other factor, or factors, are involved, over and above a direct increase in degradation rate, with increase in plasma albumin concentration, e.g. a superimposed additional catabolic rate increasing exponentially above 3 g/roe ml. No relationship was found between plasma albumin concentration and total, or e.v., albumin pool mass, or between degradation rate, and total, or e.v., pool mass. Turnover studies It is somewhat suprising that any difference at all should have emerged when the nature of the diabetic group is considered and even more that the differences should be so clear-cut. The diabetics were a very mixed collection of patients, ranging from those with newly discovered diabetes, without obvious complications, to diabetics of so-40 years standing, some with, and some without complications. Nevertheless, the turnover studies revealed one clear cut point of difference between the groups. The rate of clearance of label from the plasma was increased in the diabetics from day 2-3 onwards, the difference became greater as time passed, and it is reasonable to
272
MCPHERSON
accept that this represented clearance of labelled albumin. Albumin could leave the intravascular compartment at a greater rate by a number of different mechanisms. (a) Increased degradation: This was not demonstrated. In the 39 studies with adequate urine collections, the plasma degradation rate constant (k,), showed no difference between groups. In the smaller number in which absolute degradation rates were measured, there was also no difference. (b) Increased loss into another pool, or pools. The demonstrated overall increase in extravascular pool size reflected the tcndencv to increased total body albumin pool size in many, but not all, diabetics. Those with the largest pool sizes tended to have vascular complications, but the converse was not exclusively true. It would be reasonable to explain the turnover data by this difference, with increased rate of transfer into a larger pool, but for the observed fact that the difference between the groups continued to increase beyond equilibrium time. Equilibrium time itself was the same for the two groups. This suggested either an alternative route of loss from the plasma compartment, or that the two compartment system provided an invalid model for analysis of the turnover data. Other route of loss from i.v. comfiartvaents The model described envisaged only one route of loss from the total system. Loss in one direction into any further compartment was not compatible with this model. The most likely abnormal routes for loss of albumin from the intravascular compartment are urine, faeces or skin. Urine: Significant urinary protein loss was detected in only two patients. Removal of these two patient’s turnover data does not affect the validity of the between group differences, the mean value of QP at day 6-7 now being 24.45%, and 0.002 < $ < 0.01. Calculations showed that occasional minor incidents of proteinuria had not contributed significantly to the differences noted between control and diabetics. Faecal loss of jvotein: Isotope released from RIHSA secreted into the gastrointestinal tract is re-absorbed and excreted in the urines. If the faecal loss of albumin was significantly increased it would be expected that a significant increase in urinary radioactivity would occur, and that this would be reflected by increased values for fractional, and absolute degradation rates. This did not occur, and it is therefore most unlikely that the differences in plasma turnover are due to increased faecal loss. and the results suggested that there is Further work was undertaken using Xr-HSA, little or no increase in gastrointestinal protein loss in diabetic patients”. Skin: Loss of albumin by way of shed skin, or sweat, would not be reflected in degradation rate, and the label from the albumin would not be collected. However, prolonged in viva studies, using radioiodinated proteins, have shown that 95% or more of the isotope originally injected is recovered in the urine17, providing indirect evidence that loss from the skin is not excessive. Alternatively, there could be alterations in rate of entry and exit of albumin to the skin compartment (or subcompartment), as discussed below. Alternative model fey albumin distribution : To date calculations have been based on the assumption that the extravascular compartment was, or behaved as though it was, homogenous, although it has been known to consist of a large number of tissues and serous fluids. Rothschild et aZ.l8 showed that skin, muscle and viscera account for
ALBUMIN
over 60%
METABOLISM
IN
of extravascular
DIABETES
albumin,
273 but the assumption
that
these behave
as one
compartment is open to doubt, and reference to Fig. I of their paper suggests that there may be different equilibrium times for skin and muscle. Doubt has been thrown on this simple model by a number of workers, and a large variety of complex mathematical models have been considered and adopted, or rejected, on the basis of experimental evidence4~7~13~19.Takeda and Reeve13, and Reeve and BaileyI@, described mathematical methods for determining exchange rates between various compartments, and subcompartments, by which they were able to determine values for flow rates through the extravascular compartment. They proposed a similar overall model to that used in this paper, but albumin molecules were regarded as flowing in innumerable discrete streams through the interstitial fluids, with different rates of movement. An interesting, but unexplained, observation in Takeda and Reeve’s paper, was that, quite commonly, a small bump on the turnover curve was noted between day 4 and 8. This also occurred frequently in the present studies, and although the explanation is not obvious, it raises the possibility of reentry to the vascular pool from a pool of large mass, but slower turnover time. Reference to Fig. z of the paper by Katz et uLZo, raises the possibility that this could be carcass (which is mainly muscle). Katz et al. have published two papers which cast doubt on the value of all plasma protein tracer work published to date, and they challenge the conclusions reached by previous groupPyZ1. The validity of the conventional multi-compartmental model of albumin metabolism is questioned, and in particular they calculate that in both rat and human, RIHSA studies lead to underestimation of e.v. mass by 20--40~/~. They studied rats*O, and humansZ1. Extracts of tissue were made and albumin measured by a number of methods. In rats simultaneous RIRSA (radioiodinated rat serum albumin) studies were made in some experiments, and RIRSA was used to determine occluded blood in tissues. Disagreement with their methods could include doubts over the metabolic properties of their labelled albumin, their lack of allowance for sequestration and perivascular circulation of albumin22, and difficulty in knowing the state of the albumin they measured. The methods used were electrophoretic or immunological, and no assessment was made as to the metabolic behaviour of the substance measured. It is possible that some of the protein measured as albumin was, in fact, different from circulating albumin. It could have been of different, although similar, molecular structure, or might have been in a bound form, possibly to cell membranes or other proteins. Albumin may be found inside cells, and this would be included in Katz’ tissue extracts. Such “albumin” might be unavailable for, at least, short term turnover, and be additional to the exchangeable albumin. In concluding their second pape+, in which they reported no isotope studies, Katz et al. concluded that “total extravascular albumin and the distribution among pools determined from the plasma curve and the calculated exchange rates, have little or no physiological meaning”. The alternative interpretation is that in fact the isotope studies do indicate the physiological state, at least in the short and medium terms covered by most experimental work, and that conclusions can be reached regarding the exchangeable pools. It is possible that in the long term there is an exchange with the remaining static pool demonstrated by these workers, although the alternative is that the measured discrepancy indicates non-exchangeable, bound or functionally different albumin, or albumin like molecules. These criticisms and reservations do not alter the relevance of some of their
274
R~~PHERsoE~
findings. They demonstrated clearly that there are differences in the albumin content of different tissues, and in the exchange kinetics between circulating albumin and the extracellular fluid of muscle, skin and gut. This is in keeping with studies in rats23 and mice”* in which equilibrium was shown to occur at varying rates between plasma and different tissues, (although these papers do not agree in all respects) and also in humans18. It thus seems very likely that the described model used in the present and other papers is unsatisfactory. A reasonable conclusion that may be drawn from the above discussion is that the e.v. compartment comprises a number of sub-compartments that behave overall as one pool, if study is not too detailed, or disease states do not disturb transfer kinetics. Diabetes mellitus is a disease, or syndrome, in which disturbance of transfer across cell membranes and vascular walls is known to occur for small molecules such as glucose. It is possible that Ismail et al.’ may have observed a change of permeability at one intercompartmental boundary that may also occur elsewhere. Such a decreased rate of passage from the iv. space does not explain the excessive rate of disappearance in the longer term studies of the present series, but interference with transfer into other sub-compartments, and re-entry, may lead to the effects observed in these studies. The findings of Coles and MacDonald” do not appear relevant. A short paper by ColeP reports that a fall in serum albumin concentration could be produced by very high carbohydrate diets in a further group of six men, but that this could not be reproduced in six young women. As a greater intra-vascular mass of albumin, and greater rate of synthesis has been shown in normal men, but no difference in catabolic rate or distribution between the sexes, it is possible that Coles’ work reflects changes in synthesis. In the present studies no alteration of degradation rate was observed, and the only two patients with increased synthetic rates had considerable proteinuria. High carbohydrate diets may well alter serum albumin concentration by decreasing synthesis in normal subjects, but Coles’ findings cannot explain the metabolic changes in diabetes
mellitus.
CONCLVSIOX
If the concept of a homogenous extravascular pool is valid, or an acceptable approximation to reality in normal subjects, it is not applicable to diabetic patients. In view of the decreased plasma albumin pool and plasma concentration that can occur in patients with the nephrotic syndrome in association with an actual increase in total body albumin, intercompartmental transfer warrants further investigations. This is important in terms of therapeutics, particularly for patients with the nephrotic syndrome. If, in fact, the work described by Katz et a1.20921demonstrates the presence of a large subpool of inaccessible, or slowly exchangeable, albumin, it would be of great interest to know its function and the factors controlling its accessibility. Further study in diabetics could be of assistance in determining the primary patlmiogy at capillary and tissue level. ACKNOWLEDGEMENTS
I wish to thank the subjects
who made this study possible.
They gave their full
ALBUMIN
METABOLISM
informed
consent,
IN DIABETES
and suffered
a certain
275 amount
of discomfort
and inconvenience.
I also wish to thank my colleagues who consented to the study of patients under their care. The Hawke’s Bay Medical Research Foundation, The Royal Australasian College of Physicians, and the Bank of New Zealand made grants in support of this work, and the Hawke’s Bay Hospital Board provided laboratory space. In particular, I wish to thank Professor P. J. Scott, Auckland, for his invaluable advice and criticism. REFERENCES A. A. ISMAIL, K. KHALIFA AND K. R. MADWAR. Lancet, ii (1965) I - “I 810. B. L. COLES AND I. MACDONALD, Clin. Sci., 30 (1966) 37. 1. G. MCPHERSON, Clin. Chim. Acta, 49 (1973) 225. A. S. MCFARLAXE, Biochcm. J., 62 (1956) 135. I. G. MCPHERSON AND D. W. EVERARD, Clin. Chim. Acta. 17 (1972) 112. R. M. CAMPBELL, D. P. CUTHBERTSOX, C. M. MATHEWS AND’A. S. MCFARLANE,
Int. J. Appl.
Radiat., 1 (1965) 66. X. S. MCFARLANE AND A. KOJ, J. Clin. Invest., 49 (1970) 1903. t: E. MILHAUD, in H. PEETERS (Ed.), Protides of the Biological Fluids, 9 IO II
I*
13 14 15 16 17 18 19 20 2.1 22
Elsevier, Amsterdam, 1964, p. 280. 5. COHEN, T. FREEMAN AND A. S. MCFARLANE, C&in. Sci., 20 (1961) 161. T. PETERS, JR., Adv. Clin. Chem., 13 (1970) 37. W. L. BEEKEN, W. VOLWILER, P. D. GOLDSWORTHY, L. E. GARBY, W. E. REYNOLDS, R. STOGSDILL AND R. S. STEMLER, J. Clin. Invest., 41 (1962) 1312. P. W. DYKES, C&z. Sci., 34 (1968) 161. Y. T.+KEDA AND E. B. REEVE, J. Lab. Clin. Med., 61 (1963) 183. M. A. ROTHSCHILD, M. ORATZ AND S. S. SCHREIBER, Amev. J. Dig. Dis., ‘4 (1969) 711. F. L. RODKEY, Arch. Biochem. Biofihys., 108 (1964) 510. S. B. ANDERSEN AND N. ROSSING, Stand. J. Clin. Lab. Invest., 20 (1967) 183. P. J. SCOTT, Serum Beta-lipoproteins in Health and Disease, Thesis, University of Birmingham, 1962. M. A. ROTHSCHILD, A. BAUMANN, R. S. YALOW AND S. A. BERSON, J. Clin. Invest., 34 (1955)
1354. B. B. REEVE AND H. R. BAILEY, J. Lab. Clin. Med., 60 (1962) 925.
J. Katz, G. BONORRIS. S. GOLDEN AND A. L. SELLERS, Clin. Sci., 39 (1970) 705. J. KATZ, G. BONORRIS AID A. L. SELLERS, Clin. Sci., 39 (1970) 725. C. H. BAKER, Amer. J. Physiol., 216 (1969) 368. 23 W. C. DEWEY, Amer. J. Physiol., 197 (1959) 423. 24 A. SASSEN. A. M. REUTER, F. KENNES, M. TELIA AND C. DELCROIX, Rev. Euro+. BiOl., XVI (1971) 912. 25 B. L. COLES, Proc. Nutr. Sot., 28 (1969) 63A.
Etud. Clin.