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Original Article Original Article Influence of Serum Protein Levels on Serum Fructosamine Levels Munetada OlMOMl, Shogo MASUTA, Makoto SAKAI, Takeshi OHARA, Fumihiko HATA and Shigeaki BABA The serum fructosamine level is influenced by the serum protein level. Therefore, we investigated the relationship between serum fructosamine levels and serum total protein and albumin levels measured simultaneously in non-diabetic and diabetic patients. Significant positive correlations were found between them. A correction formula was constructed to express the serum fructosamine level when the serum total protein level was 7 g/dl or when the serum albumin level was 4 g/dl: corrected fructosamine (mmol/1) = measured fructosamine (mmol/1) + A (7 (g/dl) or 4 (g/dl) - serumtotal protein (g/dl) or serum albumin (g/dl)) where A is 0.28 for serum total protein and 0.30 for serum albumin in non-diabetic subjects; 0.29 for serum total protein and 0.35 for serum albumin in diabetic patients; and 0.29 for serum total protein and 0.31 for serum albumin in non-diabetic and diabetic subjects combined. Key words: Glycation, Glycated serum protein, Diabetes mellitus, Blood glucose control Serum fructosamine has been clinically used as mmol/1. The serum total protein, and serum an indicator of blood glucose control in the previous albumin levels were measured in the samples which two weeks. The fructosamine level is measured by were used for the measurement of serum colorimetry (1), taking advantage of the fact that fructose-lysine, which consists of glucose bound to For studies in vitro, glucose at concentrations of a lysine residue of serum protein, is reduced under 00 mg/dl, 200 mg/dl, and 300 mg/dl was added alkalineconditionsand, therefore, the level may be to humanserumalbumin at various concentrations affected by the level of serum proteins (2, 3). Thus, under sterile conditions. The mixture was then in- the serum fructosamine level should be expressed cubatedin 0.5 M sodium phosphate with 3 mM of after correction for serum protein level (4). Howey odiumazide, pH 7.45, at 37°C for 2 weeks. 1 fructo samine. et al. (5) have already constructed a correction s Serumsamples were used for the measurement formula for calculating the serum fructosamine of fructosamine. Fructosamine was measured level, taking the serum protein levels into considera- according to the method of Johnson et al. (1) by the tion. Wehave undertaken studies in vitro and in vivo fructosamine test (Hoffman La Roche, Basel). As to construct a correction formula for calculating the a primary standard, 1 -deoxy- l -morpholino-fructose serumfructosamine level in a similar manner. wasused. Twenty microliters of serum were colored with0.25 mmol/1 of nitro-blue tetrazolium at 37°C, MATERIALS AND METHODS at pH 10.35, and absorbance was measured at 550 The study was conducted with 175 non-diabetic nm using an automated analyzer (Cobas Mira, subjects with fasting plasma glucose ^ 100 mg/dl Japan Roche, Tokyo). and 155 diabetic patients with fasting blood glucose Serum glucose was measured by the glucose ^ 120 mg/dl and serum fructosamine levels < 4.0 oxidase method and HbAlc was measured by high- From The Second Department of Internal Medicine, Kobe University School of Medicine, Kobe, Japan Received for publication October ll, 1988. eprint request to: Munetada Oimomi MD, The Second Department of Internal Medicine, R Kobe University School of Medicine, 5-1, 7-chome, Kusunoki-cho, Chuo-ku, Kobe, 650, Japan 312 Jpn J Med Vol 28, No 3 (May, June 1989) Serum Fructosamine Assay performanceliquid chromatography (HPLC) using level and serum albumin level were significantly and the column chromatography system Auto Alc positively correlated with the serum fructosamine (Kyoto Daiichi Co., Ltd., Kyoto, Japan). level (Y = 0.285X + 0.850 andY = 0.304X + Serum total protein and serum albumin were 1.598, respectively, where Y = serum fructosamine measuredby the Biuret method and by the brom- level, X = serum total protein level or serum cresol-purple method (TBS-80S; Toshiba Co., albumin level (Fig. 1). Therefore, the corrected value Tokyo), respectively. of serum fructosamine can be expressed as follows: To test differences between correlation co- corrected value (mmol/1) = measured value efficients,normal approximation after z-transfor- (mmol/1) -A(7 (g/dl) or 4 (g/dl) - serum total mation ofStudent's t test was used. protein (g/dl) or albumin (g/dl)) where A is the gradient of the regression line, and 7 and 4 are the RESULT S target values of the corrected serum total protein and In non-diabetic subjects, the serum total protein Table 1. Fructosamine correction formula 4 .0 C or re c ted fructosamine = (m ea s u re d f ru ct o sa m in e + A (7.0 o r 4.0- s er um to ta l p ro te in N = 1 7 5 m m o l/i? m m o l / f i o r se ru m albu m in co n c e n tra tio n , a /d JG)) m m o l/ ｣ Y = 0 . 2 8 5 X + 0 . 8 5 0 R = 0. 6 3 1 A P < 0 . 0 0 1 3 .0 s e r u m t o t a l p r o t e in s e r u m a lb u m in CD c : ! ; ii ｣ fl N o n - d fb jeecttics o .2 8 0 .3 0 s u I/) o 3 2 .0 D ia b e t ic p 13 p a t ie n t s 0 .2 9 0 .3 5 en0) ( F r u c t o s a m i <n e4 .0 ) 1. 0 A l l s u b j e c t s ( F r u c t o s a m i 0n. e2 <9 4 . 0 ) 0 .3 1 ' 5 6 7 8 9 s e ru m t o t a l p ro t e in S /d j2 (m m o l/｣ ) G lu c o s e 4.C N = 1 7 5 Tim o l/ JO Y = 0. 3 0 4 X + 1. 5 9 8 3 .G R = 0. 6 1 9 P < 0 . 0 0 1 3 .0 . : . : l . ! i ; ! ' ! : l i: c ｣ r f i T l l - fl I/} o <D c 3 0 0 m g /d jO u e 2 . 0 I 2 .0 ra l/> e o G lu c o s e 3 u CD 3 2 0 0 m g /d jO en 1. 0 L . G lu c o s e 1 .0 1 0 0 m g /d jg 0 1 2 3 4 5 s e r u m a lb u m in 9 / d jG G lu c o s e O m g /d ｣ Fig. 1. Correlation between serum total protein and serum albumin concentrations and serum fructosamine con- 1 . 0 2 .0 3 . 0 4 .0 5 .0 centrationin non-diabetic subjects. A lb u m in (g /d jg ) Upper panel: Correlation between serum total protein and serum fructosamine concentrations Fig. 2. Fructosamine levels after incubation of various Bottom panel: Correlation between serum albumin and concentrations of human serum albumin with glucose at 37°C serum fructosamine concentrations for2 weeks. Jpn J Med Vol 28, No 3 (May, June 1989) 313 Oimomi et al m m o l/ j2 m m o l/ JZ N = 3 3 0 Y = 0 .2 94 X 4 -0 .9 37 ^ 蝣 1 Y = 0 . 2 9 1 X + 0 . 8 8 0 4 .O h 4. 0 R = 0 . 5 2 0 c P < 0 .00 1 蝣 e P < 0 .0 0 1 ra <u in c o E � * �* * ! *� * �� (0 o <n *. � f ; t .*** �'* * ｣ 3 .0 o I S 3 .0 : " . ::M H :ii i !3 -J t_ f r t> rt l i : *.:< � �� � ' : : -J H i f u -i " �� QJ (/) e 3 2 . 0 h <D 0 ) 2. 0 5T L 9 0 5 6 7 8 9 S e r u m t o ta l p r o te in g / d jg S e r u m t o ta l p r o t e in % m m m o l/ j2 m m o l/ J2 N = 3 3 0 .o Y = 0 .354 X + 1.624R=0.484 Y=0.30 8X +1. 6 7 6 4 .0 R = 0 .4 8 9 6 P < 0 .0 0 1 c en i P < 0 . 0 0 1 o fl If) o . ｫ ..' � �' . : ..� o o 蝣 :- -3 --_. ｣ 3 .0 ｣E 3.0 蝣 ｫ � �� tｫ � .1 ��iZ ^# �%^r T.*ｻ � 3 6 (U Z3 (/) <D 2.0 CO 2 .0 . �: (. �. : ! � " � ' I n 5 n o ' j i i } 0 9 /d je 1 2 3 4 5 S e r u m a lb u m in a /d jS S e r u m a lb u m in Fig. 3. Correlation between serum total protein and serum albumin concentrations and serum fructosamine con- Fig. 4. Correlation between serum total protein and serum centration in diabetic patients. albumin concentrations and serum fructosamine concentra- pper panel: Correlation between serum total protein and tion in non-diabetic and diabetic subjects. serum fructosamine concentrations pper panel: Correlation between serum total protein and ottom panel: Correlation between serum albumin and serum fructosamine concentrations U B serum fructosamine concentrations ottom panel: Correlation between serum albumin and U B serumfructosamine concentrations he corrected serum albumin, respectively (Table 1). In vitro, incubation of various concentrations of serum total protein and serum albumin levels were human serum albumin with varying concentrations significantly and positively correlated with the serum fructosamine levels (Y = 0.291X + 0.880, n=33O, oft glucose showed that the levels of fructosamine increased in proportion to the concentration of r=0.52, p<0.001 and Y = 0.308X + 1.676, albumin (Fig.
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