human α1-microglobulin: its measurement and clinical significance

Journal of Clinical Laboratory Analysis 4:376-384 (1 990)

Human a,-Microglobulin: Its Measurement and Clinical Significance

Yoshihisa ltoh and Tadashi Kawai Department of Clinical Pathology, Jichi Medical School, Minami-Kawachi-Machi, Tochigi-Ken, Japan

al-Microglobulin (a,-M), also called protein HC, is a low-molecular-weight (LMW) glycoprotein (about 30 kDa) with unique physicochemical properties. Using purified urinary al-M as a standard and specific antibody against al-M, an assay system for al-M was developed, and the clinical significance of this protein was investigated by measuring total levels of al-M under physiological and pathological conditions. al-M is distributed in various body fluids: in serum, it consists mainly of free LMW a,-M and monomeric IgA-al-M complex. The total al-M level in serum and urine

usually reflects LMW al-M variation sensitively, and its determination is quite useful as an indicator of renal glomerulotubular dysfunction and hepatic dysfunction. Serum levels can vary, depending on IgA-a,-M complex level, in parallel with the IgA concentration. The heterogeneity of a,-M purified from different sources of urine by different procedures and underestimation of IgA-al-M complex by solid-phase antibody assays can be important causes for the discrepancy of serum levels between assays.

Key words: a,-Microglobulin, protein HC, IgA, creatinine clearance, hepatoma

INTRODUCTION

Human al-microglobulin (al-M) is a brown-colored glycoprotein of about 30 kDa initially isolated from urine of patients with renal tubular disorders and biochemically char- acterized by Ekstrom et al. (1,2) (Table 1 ) . Tejler et al. subsequently purified a protein from normal urine and called it human complex-forming protein heterogeneous in charge (protein HC) since it makes a complex with IgA and albumin in serum (3). This protein has been thought to be almost iden- tical to a l -M in its physicochemical properties, structure, and function (4-6). According to Kaumeyer et al., the messen- ger RNA for a proteinase inhibitor related to the HI-30 domain of inter-a-trypsin inhibitor also encodes al-M, and the amino acid sequences of al-M consist of 205 residues, probably including 19 signal sequences (6).

al-M is distributed in various body fluids such as serum,

TABLE 1. al-M Properties (2)

PrODertV Value

Molecular weight By SDS-PAGE 31 .Ooo By gel chromatography in 6 M guanidine HCI By sedimentalion and diffusion coefficient

E at 280 nm

24.800 26.100

Carbohydrate content 19.5% 4.72 X lO"M-'cm-'

Pl 4.3-4.8 Electrophoretic mobility a1

Other properties include making dimers or polymers, binding with chromo- phore material, and making a complex with IgA.

0 1990 Wiley-Liss, Inc.

urine, cerebrospinal fluid, and amniotic fluid (7-9). In serum, al-M consists mainly of the low-molecular-weight free form (LMW al-M) and the high-molecular-weight HMW form, mainly IgA-al-M complex at varied ratios (10).

LMW al-M is easily filtered through the basement mem- brane of glomeruli and catabolized in renal proximal tubules in the same manner as other LMW proteins (1 1). Its synthetic site is thought to be mainly in the liver because of the demonstration of LMW al-M in the supernatant fluid of hepatoma cell lines (12,13). In contrast, the synthetic site and catabolic site are still unclear for IgA-al-M complex. In the structure of IgA-aI-M complex, a l -M covalently, and also to some degree noncovalently, binds to a single a-chain of theFcportion (14,15).

In 1978, we purified al-M from pooled urine from patients with renal tubular disorders, prepared its specific antibodies, and developed various assay systems with which the clinical significance of this protein was extensively studied in various body fluids by measuring levels of total a , -M (8,9,11).

Based on our original data, the clinical significance of a l -M measurement is summarized here, especially with relevance to the variations in the contribution of free LMW al-M and IgA-al-M complex.

Received February 13, 1990; accepted February 22, 1990.

Address reprint requests to Yoshihisa Itoh, M.D. , Department of Clinical Pathology, Jichi Medical School, Minami-Kawachi-Machi, Tochigi-Ken, Japan.

Human u,-Microglobulin 377

THE ESTABLISHED ASSAY SYSTEMS FOR CY~-M

To date, four different quantitative assays and one qualitative method have been developed for the measurement and detection of total a l-M; single-radial immunodiffusion (SRID) , an enzyme-linked immunosorbent assay (ELISA), a double- antibody radioimmunoassay (RIA), a solid-phase RIA, a latex aggulutination photometric immunoassay (LAPI) using an automated instrument (LA-2OO0, Eiken, Japan), and a manual latex agglutination test (LAT) (18,19,21). Although its clinical application may be limited, an ELISA for IgA-al-M complex has been established (22,23). With the introduction of these assay systems, the clinical study of al-M has been further facilitated and the measurement of this protein is now a routine procedure in many clinical laboratories in Japan. The assay systems established so far are discussed below.

DEVELOPMENT OF al-M ASSAY SYSTEMS

Preparation of Purified al-M and Its Antibody

Purified a l -M used as a standard was initially obtained from pooled urine from patients with renal tubular disorders in a manner similar to that of Svensson and Ravnskov (16). Modifying an original procedure, we have finally established our present purification steps with high yield (about 20%) and high purity (over 95%), which is described else- where (17). In brief, pooled urine from patients with chronic renal failure (CRF) was precipitated with 40% ammonium sulfate and its supernatant was further saturated to 75% (step 1). After dialysis, ct-M-rich solution was fractionated by column chromatography in four steps: a concavalin-A (con-A) affinity column (step 2), gel-filtration (step 3), an anion exchange column (step 4), and an anti-urine protein rabbit antibody coupled affinity column (step 5). Purity was confirmed by sodium dodecyl sulfate polyacrylamide gel elec- trophoresis (SDS-PAGE) (Fig. 1) and immunoelectrophoresis (IEP).

The standard was prepared after measurement by optical density at 280 nm, assuming E = 17.288, as reported by Ekstrom et al. (1). This purified al-M has been used as the standard for assays in Japan.

Specific antibodies against al-M were prepared by inject- ing purified protein into goats and rabbits, and the antibodies were purified by an anion exchange chromatography. Their specificity were confirmed by IEP and Ouchterlony immunodiffusion ( 18).

SRID

First, 10 ~1 of serum is placed into wells in 1% agarose gel containing 1.5% goat anti-al-M antibody and incubated at room temperature for 48 hr. The diameter of the precipitin ring thus formed is measured, and the concentration in samples is obtained from the standard curve. Depending on the affinity and avidity of antibody used, this method can detect serum levels ranging from 6 to 96 mg/L without sample dilution. Since its sensitivity coincidentally corresponds with the upper limit of normal in urine (10 mg/L), this method is a simple and cost-effective urinary test for screening for glomerulotubular dysfunction (24).

Fig. 1. Purification of a,-M from pooled urine. I: precipitates by ammonium sulfate. 11: con-A-bound fraction (Fr). 111: Fr obtained by gel-filtration. IV: Fr obtained by anion-exchange columns. V: purified a , - M obtained from

a final step of an anti-urine protein affinity column. A single band was observed showing a molecular weight of purified a,-M at about 30 kDa in a final step V.

378 ltoh and Kawai

ELSA

We used a sandwich method for the enzyme immunoassay of a,-M. The first reaction occurs between al-M in samples or standards and anti-aI-M goat IgG coated on a polystyrene bead. In the second step, peroxidase-labeled anti-al-M goat IgG is reacted. Finally, an enzyme-substrate reaction is fol- lowed. The amount of antigen sandwiched between antibody on the bead and peroxidase-labeled antibody is reflected in the degree of enzymatic activity. The level of a]-M is esti- mated from the standard curve of enzymatic activity.

The procedure is nonisotopic and relatively simple, measuring concentrations ranging from 15 to 480 ng/ml. Based on this assay, a quick and convenient new commercial assay system has been developed (Irnmunozyme, Fuji Rebio, Tokyo, Japan) (22).

RIA

A double-antibody RIA is at present one of the most pop- ular methods employed in Japan. A competitive reaction occurs between 12%al-M and al-M in standards or samples to rabbit anti-al-M antibody in free fluids (first reaction). The antirabbit IgG goat antibody is reacted with the al-M rabbit IgG complex (second reaction) and then centrifuged. The radioactivity of the precipitates is measured by a gamma counter. This procedure is quite simple, taking 4 hr from sample preparation to the determination of the al-M level. It can measure concentrations ranging from 25 to 800 ng/ml. Re- cently, a modified kit became commercially available without sample dilution, thus reducing assay time to 2.5 hr (AMG RIA, Shionogi, Osaka, Japan).

LAPI

LAPI is a recently developed automated assay capable of measuring 200 samples within 2 hr. The turbidity of latex agglutination is measured at 585 nm, at which point the variation of degree of rabbit anti-a,-M antibody-coated latex agglutination (absorbance) is in proportion to the quantity of al-M (Fig. 2). This LAPI can determine serum levels ranging from 3 to 98.5 mg/L and urinary levels from 0.5 to 16.8 mg/L. A prozone can be detected by either unusually high or unusually low absorbance at an initial reaction, and sample dilution can be easily adjusted to fall within an antigen- antibody optimal ratio.

Manual LAT

Polystyrene latex particles (0.22 nm in diameter) are coated with anti-at-M goat IgG according to an established method (25), then 100 p1 of test samples properly diluted with 0.01 M phosphate buffer containing 0.4% bovine serum albumin is placed on a glass slide and 50 p1 of anti-al-M-coated latex solution is mixed reacting for 3 min. The degree of latex agglutination is observed with the naked eye. The procedure is a

quite easy method to detect the presence of al-M in biologi- cal fluids. The sensitivity of this LAT was about 100 ng/ml.

INTERLABORATORY VARIATION OF SERUM a1-M LEVELS

The normal serum level of al-M has been reported by various investigators to be between 10 and 100 mg/L. Grubb et al. clearly demonstrated that this variation is due to the measurement of serum IgA-al-M complex (HMW al-M) by assays that use LMW al-M as a standard (14). A subsequent study has shown that the purified material had a 34 kDa in molecular weight by a different purification procedure (26). Even with the same procedure as ours, a protein of 25 kDa was obtained from a single urine and immunochemically identified as a ]-M ( 1 7). The difference in serum levels among assays resides in the heterogeneity of the al-M standard and its antibody in each assay system.

BETWEEN-METHOD VARIATION OF SERUM ~t1-M LEVELS

Using the same purified antigen of 30 kDa and the same rabbit antibody, three different assay systems were set up in an attempt to define precise mechanisms (17).

Although urinary levels showed almost no difference among assays, a discrepancy was noted in serum, especially in cases with IgA multiple myeloma (MM), in which the average level was two times higher by a double-antibody RIA than by two solid-phase antibody assay systems, such as ELISA and LAPI (Fig. 3). Sera from patients with IgA MM was fractionated by gel-filtration, and the level of each fraction obtained was comparatively measured by RIA and ELISA. Two main al-M- positive peaks were obtained; one was an IgA-al-M-rich peak in a slightly earlier peak of monomeric IgA and the other was LMW al-M. In the IgA-al-M-rich fraction, the value by RIA was about three times higher than that by ELISA, while the values in the LMW aI-M fractions were the same. Because of its steric hindrance, the IgA-al-M complex was more weakly bound to solid-phase antibody than was the same antibody in free fluid in RIA. The binding of IgA- orl-M complex was further blocked by the LMW a]-M con- comitantly present in serum, as was tested by a reconstitu- tion study by adding purified LMW a]-M in ELISA for IgA-al-M complex (27). It is therefore suggested that underestimation of IgA-al-M complex by solid-phase antibody assays is another important cause of the discrepancy in serum levels among assays.

Urinary levels determined by different assays were without significant differences because the a ] -M present in urine is, for most part, of LMW. No IgA-al-M complex was identified in urine ( 10).

Serum values of al-M can vary more or less according to the assay system used because of the influence of IgA- aI-M complex. The establishment of strict assay systems for

Human u,-Microglobulin 379

Latex Solution Sample Application Mixing Caribration

:

. . 400p1

10 Sec --+

10 Sec 3

Diluted 10 times with 0.2M

30 Sec c T2

Measured at 585nm

-+ Discard

‘x

Q) Sample Application T A Absorbance m 5 0 v) n Q !

Time

Rg. 2. A schematic presentation of a latex agglutination photometric immunoassay (LAPI) (courtesy of Eiken Co. , Ltd, Tokyo, Japan).

separate measurement of each form would be ideal, using a monoclonal antibody recognizing an epitope of each form spe- cifically and discriminatively. However, it is LMW al-M that contributes to the dynamic variation of total serum and urinary values in most clinical conditions, and any single method can be used for routine clinical evaluation. Double-antibody RIA and SRID, competitive assays in basic principles, seem to offer reliable results in case with marked IgA elevation.

CLINICAL APPLICATIONS OF a1-M

Sampling

There were no significant differences in the al -M levels between serum and plasma as tested by ELISA (unpublished data).

The stability of a , - M was investigated by comparing it with that of P2-microglobulin (P2-M) (Fig. 4). Serum and urine

were stored at 25°C and 4°C under three different pH conditions, and al-M and P2-M levels were determined on four consecutive days by RIA. In serum, both a , - M and P2-M were quite stable at 4°C and 25°C at all three pH conditions. In urine, al-M is stable at 4°C at all three conditions. al-M was also stable in urine at pH 5 and pH 6 at 25°C. In contrast, urinary P2-M degraded instantly at 25°C at low pH. Degre- dation also occurred in purified al -M and P2-M when they were added to serum under the same conditions. The precise mechanisms are unknown, but urinary acid proteases seem to play an important role in the degradation of proteins, while the structure of al-M makes it resistent to enzymatic digestion. Because of relatively weak protease activity in serum, both a , - M and P2-M may be unchanged in serum. They may be protected by protein-protein interactions at a high total protein concentration.

380 ltoh and Kawai

Serum

Normal

CRF

~

a,-m Level (mg/I) N (Meanf 1 SD)

RIA <o.o 1 6 <0.05

50 18.5f4.9 16.5&-3.6 13.0f2.3

58 78.6f57.5 64.7f47.2 70.2f42.3 p<o.o 1 I I I" p<o.o 1 I I

IgA MM Urlne

CRF

50 41.6rt19.7 20.349.4 19.6f10.7

20 15.lfll.O 13.6f11.9 13.0f10.6

?# Slgnlflcant dlffrrrnce

1 I:----"-

Fig. 3. Discrepancies in serum a , - M concentrations in our assays (17). The same standard and antibody were used throughout.

1 I:----"-

When serum and urine were stored at - 20"C, it proved to be stable at least 1 yr and at 4°C for 1 mo. Three cycles of freezing and thawing had no effect on its stability for both fresh and stored samples at - 20°C for 1 yr.

When purified a, -M or a , -M present in urine was stored at 4°C for more than a month, uI-M gradually polymerized. Polymer formation in samples may cause underestimation of the sample level and overestimation of the standard. One must always be cautious about the storage condition of samples.

Normal al-M Concentrations

Using SRID, ELISA, and RIA, the distribution of a l -M levels in normal individuals was extensively studied; al-M was found to be present in various body fluids. No al -M was, however, detected in feces, gastric juice, or bile (Table 2) (8,9).

Physiological Variations of Serum al-M

No diurnal variation was found in healthy individuals with no restrictions on their daily living habits (29). Also, no significant increase was observed in the plasma level of a,-M at any time after strenuous physical exercise (bicycle ergome- ter, about 75% v 0 2 max for 30 min) (30).

A significant difference was seen in the serum level of al-M between males and females at all ages, being higher in the

Serum A- pm atQC - prrn a t 2 f C

m-m NOrrnaL aubJacta (pg/ml) . Puttants (pg/mO

50 c J 10.0 I-

0 1 2 3 4 0 1 2 3 4

*(n/mu Time interval (days)

0 1 2 3 4

0 1 2 3 4

100

50

pH5.0 976 I

50 'i 0 1 2 3 4

Time intervat (days)

Fig. 4. Stability of a ] - M and P2-M in serum and urine under different pH and temperature conditions.

Human a,-Microglobulin 381

Furthermore, LMW al-M has been identified in two different hepatoma cell lines (12,13). These findings indicate that LMW al-M is synthesized mainly in the liver, and its serum decrease may sensitively reflect the lowered synthetic capacity of liver parenchymatous cells since LMW a,-M is rapidly cleared from the plasma by filtration through the glomeruli.

The formation of IgA-al-M complex seems to be slight in liver disease. In 37 cases of liver disease with increased levels of IgA, only one had a raised al-M level. The increased IgA is mainly of dimeric form, which does not appear to bind

Despite liver cirrhosis as a complication of hepatoma, a l -M values in hepatoma were significantly higher than in liver cirrhosis; furthermore, there was a case with marked elevation of al-M. These cases had no renal complications as shown by normal serum creatinine (Cr). The elevation was probably due to increased a]-M synthesis and secretion by hepatoma cells, exceeding the clearance capacity of the kidney (31). The protein produced by hepatoma cells seems to be structually different from that in normal and other pathological conditions. The assay using a monoclonal antibody that recog- nized the epitope of al-M on the IgA complex has detected serum increases in 35 out of 77 cases with hepatoma (13).

Urinary excretion of a l -M can be low in liver cirrhosis, reflecting the decreased serum level (31).

al-M (15).

Chronic persistent

Chronic aggressive

Chronic aggressive

hepatitis

hepatitis ( I l A )

hepatitis ( I lB)

Liver cirrhosis (compensated)

Liver cirrhosis (decornpensated)

TABLE 2. Distribution of a l - M in Body Fluids

Sample Method al-M concentration

I I

. d . . . I I I

I

I

I . . I . . . I

I

; .. ... : : I I&---

. , .. . ..... . . I I

I I

Y ..... I . .... . I'l I

Serum SRID 18.9 ? 5.6 mg/L Umbilical cord blood ELISA 10.4 2 3.7 mg/L Urine RIA 2.14 ? 1.20mg/day Cerebrospinal fluids ELISA 34.8 ? 16.0pg/L Amniotic fluids ELISA 2.65 ? 0.17 mg/L Pleural fluids* RIA 6.2 2 4.3 mg/L Ascitic fluids* RIA 4.8 2 1 .8mdL

Values are means f 1 SD. *Obtained from patients.

male than in the female. a l -M values are also higher in the elderly (8). The precise reasons for the varying levels are unknown. As can be seen in P2-M, variations may be the result of physiological changes in renal function. The physiological elevation of IgA can be another important factor influ- encing age and sex differences.

In pregnancy, serum al-M reaches a peak at full-term and returns to normal postpartum (9).

Pathological Variation of a,-M Levels

Hepatic diseases

Serum levels of al-M were measured by SRID in 78 patients with clinically well-defined hepatic diseases (Fig. 5) (31). The serum level was considerably decreased in liver cirrhosis and fulminant hepatitis. Except in hepatoma, there were significant correlations between the level of a l-M and albumin (r = 0.56; p < 0.01), fibrinogen (r = 0.53; p < 0.01), and cholinesterase activity (r = 0.48; p < 0.01). Recent chromatographic analysis has demonstrated that serum LMW al-M was markedly decreased after hepatectomy (unpublished data).

10 20 30 40 I

I

I . Acute hepatitis I 1 . .. . Fulrninant .. heoatitis I

I

I- " I . . .'. I .. : .. .. .I.. . Hepatoma . . Fig. 5. Serum variations in al -M in hepatic diseases.

Renal disease

By using SRID for serum and ELISA for urine, variations were studied in 59 patients with glomerulonephropathy. The serum level was elevated in significant correlation with Cr, BUN, and P2-M (r = 0.70; p < 0.001). It was also inversely correlated with Cr clearance (Ccr), a rough indicator of glomerular filtration rate (GFR) (Fig. 6). Serum al-M started to increase over a normal range when Ccr fell below 80 L/day, while serum P2-M and Cr were within normal ranges. This result was due to different handling of al-M with other param- eters in association with its physicochemical properties such as a relatively large molecular weight, isoelectric point (PI 4.5-4.8), and elongated molecular shape. Urinary a l -M excretion was also elevated in the patients in whom Ccr was within normal limits. When Ccr became less than 80 l/day, al-M urinary excretion became elevated to a marked degree, then reaching a plateau. Provided the renal disease was not complicated by either severe hepatic dysfunction or a marked increase of serum IgA, the combined determination of total serum and urinary al-M was a useful tool for the detection of mild reductions of GFR (1 1). The variation of serum a]-M in renal diseases is mostly of LMW al-M, while serum IgA-al-M complex is unchanged except in some cases with IgA nephropathy (27).

a l -M has been established as a diagnostic and prognostic aid for the clinical assessment of renal tubular dysfunction. Like other LMW proteins, its urinary excretion was elevated

382 ltoh and Kawai

10 -

- 5 - F F

I .r

. log Cr =1.51-0.70 x log Ccr

I .-0.94 n :59 p c 0.001

log aI m=2.30-0.42x log Ccr

r =-0.74

F

5 E’ -

10

1 5 10 50 100 24-haw creatinine clearance, llday

1 log p2 m = 2.06-0.91 x log Cc,

n =41 p .c 0001

1 S io 2C-hour creatlnine clearance, llday

.e I \;.. c

b 1

+- 0,

5 9i 05 ! I I I I

1 5 l o 50 100 24-hour creatinine clearance, llday

Fig. 6. Serum a,-M value and creatinine clearance in renal disorders.

in patients with cadmium poisoning and Balkan nephropathy and well correlated with P2-M and retinol binding protein (24,32). Urine protein profiles after bum injury were initially of a mixed glomerular and tubular type, gradually changing to tubular proteinuria. Urinary LMW protein excretion gen- erally changed in parallel. However, P2-M and retinol binding protein returned to normal in 2 wk after bum injury, while a l -M excretion remained at a high level for several weeks (33).

Based on these findings, renal handling of al-M was ana- lyzed. In a patient with advanced cadmium poisoning with selective renal tubular dysfunction, the urinary excretion of al-M per day was 94 mg, which was almost equivalent to the maximal filtered load to tubules as judged by the fact that P2-M excretion was 100 mg/day+lose to its maximal filtered load (34). Provided LMW al-M is present at the level of 10 mg/L in serum and GFR is about 170 Wday, the glomerular sieving coefficient (GSC) is thus calculated at 5.5%. Urinary excretion of al-M in normal individuals is about 3 mg/day. Only about 3% of filtered al-M is, therefore, excreted in urine, and the other 97% is reabsorbed and catabolized in proximal tubules.

As compared with P2-M, al-M is quite stable in urine, relatively free of prerenal variation, and present in high enough concentrations to detect abnormal elevation by SRID. More- over, the determination of its level in serum and urine sensitivity detects decreased GFR in early stages. In view of these properties, al-M has become established as a novel indicator of glomerular and tubular function.

Serum a,-M and IgA in various diseases

A marked elevation of serum total a l -M value was observed in patients with IgA MM in the absence of impaired renal function. Although no direct correlation was present between the serum orl-M and IgA concentrations, an IgA-dependent increase of al-M was noted (27), and in those patients with an IgA level over 1 ,OOO mg/dl, the total al-M level was raised above the normal upper limit of 30 mg/L. Neither IgA sub- class nor light chain type predominance was shown to influence the cx -M concentration.

Crossed-intermediate-gel IEP qualitatively showed an elevation of monoclonal IgA-bound al-M (IgA-a l-M complex)

Anti a l-rn antlbody t

L c 0 c

Antl-a l-rn antibody

Antl-lgA antibody

-- 1 5 V / c m tor 6Omln

Fig. 7. Demonstration of IgA-bound a,-M (IgA-a,-M complex) (arrow) in the p region by intermediate-gel crossed immunoelectrophoresis.

Human a,-Microglobulin 383

TABLE 3. Serum and Urinary a l - M Variations Related to LMW al -M and IgA-al-M Complex

Total Serum

u,-M concentration LMW -1-M IgA-al-M complex Urine LMW a,-M

Elevated Renal dysfunction; Monoclonal and polyclonal Renal dysfunction; tubular, decreased GFR increase of IgA glomerular, and mixed

overproduction (?)

severe liver cell damage

Hepatoma (rare);

Decreased Hepatic dysfunction; IgA deficiency Hepatic dysfunction (some cases)

Infection with increased IgA

in the p region (Fig. 7); the increase of IgA-al-M complex was directly demonstrated by an ELISA (22,23,27) (Fig. 8).

Polyclonal increase of IgA is also a cause of raised serum al-M. Vincent et al. found that IgA-al-M complex was elevated in IgA nephropathy (35), in which disease localization of al-M in the mesangium was immunochemically demonstrated by Murakami et al. (36). In immunodeficiency, the total a l -M level was about half of that in healthy individuals, reflecting marked decrease of IgA-al-M complex (Fig. 8).

Extensive studies have been carried out to identify the synthetic site of IgA-al-M complex. No al-M has been identified immunochemically in plasma cell lines and plasma cells in bone marrow aspirates with IgA myeloma. Synthesis of IgA-al-M complex is unlikely to occur in plasma cells. Mono- meric IgA, once released from plasma cells, may bind with LMW al-M covalently in some other tissues.

The variations in serum and urinary al-M are summarized in Table 3. LMW al-M sensitively reflects hepatic dysfunction and renal dysfunction and contributes to the dynamic change of total level of al-M. IgA-al-M complex also plays an important role in total al-M, especially in cases with increased IgA.

Fifteen years have passed since the initial isolation of this protein from urine, and major contributions have been made to the knowledge of its physiochemical properties, structure,

. . . . ' ' .* . '

IgA-ai-rn Complex ( n g l m l ) '

10 20 30 40 50 60 70 80 90

t : Expressed in u i - m Level

Fig. 8. IgA-al-M complex levels in immunoglobinopathies. The concentrations were determined by an ELISA (27).

origin, and fate. With the development of fundamental studies, the clinical application of al-M assays has been expanded, leading to further investigations. However, much uncertainty still remains about the role of al-M in clinical chemistry. In the next few years, the continued study of a l -M is expected to resolve some of these uncertainties.

ACKNOWLEDGMENTS

This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education (Project Number 62480435) and a Research Grant from Tochigi Association of Preventive Nephrology. The authors are grateful to Dr. K. Takagi, Dr. K. Kin, and Dr. H. Enomoto for their distin- guished contribution to the study of this protein. This paper was kindly reviewed by Emeritus Professor E.H. Cooper, School of Medicine, University of Leeds, U.K.

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human α1-microglobulin: its measurement and clinical significance

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