An automated assay for measuring serum ascorbic acid with use of 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy, free radical and o-phenylenediamine.

We developed a novel, cost-effective, and automated assay for ascorbic acid (AsA) in serum using a COBAS MIRA S analyzer (Roche Diagnostic System). Our method has a wide dynamic range and covers AsA concentrations from well below the lower reference interval to well above it. AsA is oxidized by 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy, free radical (TEMPO) to dehydroascorbic acid (DAsA). The latter condenses with o-phenylenediamine (OPDA) to form a quinoxaline derivative that absorbs light at 340 nm. The change in absorbance at 340 nm is proportional to the concentration of AsA in the specimen. The automated system permitted the assay of 65 specimens per hour at a cost of approximately US$ 0.01 per specimen for reagents. The assay can be applied directly to serum specimens (direct method) and also to sera with a prior deproteinization step with metaphosphoric acid. The detection limit for the direct serum assays is 0.8 vs. 0.4 mg/l with the deproteinization method. The recovery of AsA from a supplemented serum pool was of >95% for both procedures. We used four distinct methods on 66 patients sera. The direct method for AsA correlated well with an HPLC method (r=0.964, P<0.001); the direct method also correlated well with a method that uses AsA oxidase (r=0.975, P<0. 001). The deproteinization method correlated well with HPLC (r=0.981, P<0.001), and with the AsA oxidase procedure (r=0.994, P<0.001). Ten within-day determinations on a serum pool gave a C.V. <4.3% for both the direct and deproteinization procedures. The between-day assays of the same serum pool over 10 days gave a C.V. of <6.7% by both methods.

[Serum amyloid A (SAA) 1, SAA 2 and apolipoprotein E isotype frequencies in rheumatoid arthritis patients with AA amyloidosis].

OBJECTIVES: To examine the relationship between polymorphism of serum amyloid A (SAA) 1, SAA 2 and Apolipoprotein E (Apo E) and susceptibility to AA amyloidosis (AA) in rheumatoid arthritis (RA). METHODS: We compared the frequencies of SAA 1 alleles (alpha, beta, gamma), SAA 2 alleles (alpha, beta) and apo E alleles (epsilon 2, epsilon 3, epsilon 4) in AA-positive RA with those in AA-negative RA. Each isotype was analyzed by the following method: SAA 1 and SAA 2 by PCR-RFLP and Apo E by Western blotting method. Blood samples were obtained from 50 AA-positive RA patients with SAA 1 isotype, 50 AA-negative RA patients with SAA 1 isotype, 27 AA-positive RA patients with SAA 2 isotype, and 26 AA-negative RA patients with SAA 2 isotype, respectively. Likewise, Apo E isotype was determined by withdrawing blood samples from 61 AA-positive RA cases and 51 AA-negative RA cases. RESULTS: In AA-positive RA, each frequency of three different alleles of SAA 1, i.e., alpha, beta and gamma was 15%, 32% and 53%, while it was 32%, 28% and 40% in AA-negative RA. The allelic distribution between AA-positive RA group and AA-negative RA group was significantly different (P = 0.00163) with a lower frequency of alpha allele and a higher gamma allele frequency observed in AA-positive RA group. The frequency of each SAA 2 alleles (alpha & beta) was almost identical: 88.9% and 11.1% in AA-positive RA versus 90.4% and 9.6% in AA-negative RA with p value of 0.8007. Each frequency of three different Apo E alleles (epsilon 2, epsilon 3 & epsilon 4) was 4.9%, 85.2% and 9.8% in AA-positive RA, while in AA-negative RA it was 7.8%, 86.3% and 5.9%, respectively. The AA-positive RA group showed a slightly higher prevalence of epsilon 4 allele than the AA-negative RA group, yet the difference did not reach statistical significance (P = 0.3969). CONCLUSIONS: These results suggest the possibilities that SAA 1 alpha may be working protectively against and SAA 1 gamma provocatively for the development of AA amyloidosis in RA. However, there was no significant association between SAA 2 isotype patterns and the development of AA amyloidosis in RA. Furthermore, there was no discernible association between AA amyloidosis in RA and Apo E 4 isotype.

Intractable diarrhoea associated with secondary amyloidosis in rheumatoid arthritis.

OBJECTIVE: To examine the clinical characteristics of intractable diarrhoea associated with secondary amyloidosis in rheumatoid arthritis (RA). METHODS: Of 179 RA patients with biopsy confirmed secondary amyloidosis, 24 cases (23 women and one man) with intractable diarrhoea lasting for more than one month were retrospectively evaluated. RESULTS: The mean (SD) duration of diarrhoea was 87 (64) days. Prodromal symptoms of gastrointestinal dysfunction (n = 21) and impaired peristalsis (n = 16) were observed. Laboratory data showed hypoproteinaemia (4.7 (0.85) g/dl) caused by malabsorption or protein loss and high values of C reactive protein (17.0 (9.3) mg/dl). Recurrence of intractable diarrhoea (n = 4) and transition from intractable diarrhoea to other gastrointestinal problems of amyloidosis (ischaemic colitis (n = 2) and intestinal pseudo-obstruction (n = 4)) were observed. In 19 patients (25 episodes) the duration of intravenous hyperalimentation at remission (18 episodes) was 68 (52) days. Corticosteroid pulse therapy was administered to 10 patients (11 times) and the time elapsed from the end of corticosteroid pulse therapy to the end of diarrhoea was 18 (14) days. One and five year survival rates after the onset of intractable diarrhoea were 73.4% and 38.9%. Seven of 13 patients (54%) had died as a result of infectious diseases. CONCLUSION: Intractable diarrhoea associated with secondary amyloidosis in RA is a serious clinical entity and the prognosis is poor. Although it is assumed that intravenous hyperalimentation treatment and corticosteroid pulse therapy are favourable regimens for intractable diarrhoea, the patients should be monitored for possible infectious complications.

Hepatic cirrhosis showing false-positive serum C-reactive protein reaction.

A 58-year-old male who had been diagnosed as hepatic cirrhosis four years previously was admitted to our hospital because his serum C-reactive protein (CRP) level had gradually risen, reaching 139 mg/dl. No inflammation findings were observed subjectively or objectively. Close examination revealed his CRP reaction to be false positive. His serum CRP showed positive only in a latex agglutination method using goat anti-CRP IgG. This false-positive reaction was thought to be owing to the abnormally glycosylated IgM, which has an affinity for the goat serum IgG.

Long-term effects of delapril on renal function and urinary excretion of kallikrein, prostaglandin E2, and thromboxane B2 in hypertensive patients.

The effects of delapril, an angiotensin converting enzyme (ACE) inhibitor, on renal function and the renin-angiotensin and kallikrein-prostaglandin systems were investigated in 10 hypertensive patients who were treated for between 4 months and 1 year. There was a significant (P less than .05) increase in renal blood flow (RBF) without affecting glomerular filtration rate. Filtration fraction increased, while renal vascular resistance decreased. There were also significant (P less than .05) increases in urinary kallikrein and prostaglandin excretion, while thromboxane excretion decreased. There was no change in urinary aldosterone excretion. These results suggest that renal hemodynamic changes seen during long-term therapy with delapril are caused, in part, by activation of the kallikrein-prostaglandin system, as well as suppression of the renin-angiotensin system.

Effects of atrial natriuretic peptide on renal arterioles: morphometric analysis using microvascular casts.

In normal rat kidneys, the effect of atrial natriuretic peptide (ANP) on the diameter of the arterioles was evaluated by scanning electron microscopy of vascular casts. Acryl resin was infused into rat kidneys during the administration of ANP, either alone or with norepinephrine (NE). ANP infusion constricted the proximal efferent arteriole in the superficial cortex. Although NE constricted the proximal and distal segments of the afferent arteriole in the superficial cortex, the addition of ANP reversed the constriction and further constricted the efferent arteriole. In the deep cortex, only the proximal segment of the afferent arteriole was dilated by ANP when infused with NE. In a separate set of experiments, ANP increased both the glomerular filtration rate (GFR) and urinary sodium excretion (UNaV), and NE decreased the renal blood flow (RBF). However, administration of ANP after NE recovered RBF and increased GFR as well as UNaV. Results indicate that ANP increases GFR and natriuresis by constricting the efferent arteriole. NE appears to decrease RBF by constricting the afferent arteriole. ANP antagonizes the renal effects of NE primarily by dilating afferent arterioles.

[The long-term effects of a new converting enzyme inhibitor, delapril hydrochloride, on renal function, renin-angiotensin-aldosterone system and kallikrein-kinin prostaglandin system in hypertensive patients].

The effects of a new angiotensin converting enzyme inhibitor, delapril hydrochloride, (delapril) on renal function, and the renin-angiotensin-aldosterone and kallikrein-kinin prostaglandin systems were studied in 10 hypertensive patients. After 4 to 12 months (7.6 +/- 0.9 [SE]) of treatment with 15-60 mg/day (36 +/- 6.8) of delapril (b.i.d.), mean arterial pressure was decreased from 126 +/- 3.0 to 110 +/- 4.4 mmHg (p less than 0.01). Although renal blood flow (RBF), assessed by PAH clearance and hematocrit, was increased from 437 +/- 51 to 490 +/- 49 ml/min (p less than 0.05) and renal vascular resistance was decreased (p less than 0.05), glomerular filtration rate, measured by endogenous creatinine clearance, did not change significantly. Thus, filtration fraction was reduced (p less than 0.01). Plasma renin activity was increased from 1.5 +/- 0.3 to 4.4 +/- 1.1 ng/ml/hr (p less than 0.01). Plasma aldosterone concentration tended to decrease (p less than 0.1), and urinary aldosterone excretion showed on significant change. Although urinary kallikrein and prostaglandin E2 excretions were increased (p less than 0.05), urinary thromboxane B2 excretions was reduced (p less than 0.05). In addition, the changes in RBF were significantly correlated with those in urinary PGE2 excretion (r = 0.63, p less than 0.05). These results suggest that the antihypertensive effect of delapril is multifactorial and that the improvement of RBF seen during delapril administration in the present study may be partly due to the suppression of the renin-angiotensin-aldosterone system and the activation of kallikrein-kinin-prostaglandin system.

Factitious proteinuria in a young girl.

A 16-year-old girl showed waxing and waning proteinuria and fat globules in urine for three years. There were no other abnormal findings except for the urinalysis. The renal biopsy findings did not indicate glomerular disease. Electrophoresis of the urinary protein showed two abnormal fractions at the alpha and beta globulins. Immunoelectrophoresis demonstrated that these abnormal proteins were not derived from human serum proteins, but were egg proteins. It appears that the proteinuria was factitious and that egg proteins were injected into the bladder, as they were also present in the bladder urine.

Morphometric analysis of arteriolar diameters in experimental nephropathies: application of microvascular casts.

Renal arteriolar diameters were measured, using microvascular resin casts, in two hyperfiltration models of rats: the remnants kidney of subtotal nephrectomy (NX) and streptozotocin-induced diabetic kidney (DM). In the NX, the blood pressure was elevated, urinary protein excretion was markedly increased and glomeruli were severely damaged. In the DM, although the blood pressure remained normal, urinary protein excretion was significantly increased and glomeruli were damaged but to a lesser extent than in the NX group. In the NX group, the afferent arteriole was dilated and the efferent arteriole was constricted. In the DM group, the afferent arteriole was dilated, while the efferent arteriole remained unchanged. The results showed that afferent arteriolar dilatation was seen in both the NX and DM groups, possibly leading to the glomerular damage. In the NX group, the systemic high blood pressure and efferent arteriolar constriction augmented glomerular damage significantly.

Variations in renal arteriolar diameter in deoxycorticosterone acetate-salt hypertensive rats. A microvascular cast study.

The relation between hypertensive glomerular damage and arteriolar diameter was examined in a microvascular cast study in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. The blood pressure and urinary protein excretion increased progressively in the DOCA rats. In controls afferent arteriolar diameters increased during the course of the experiment, and efferent arteriolar diameters remained unchanged. In the DOCA rats, however, afferent arteriolar diameters did not change significantly, while efferent arteriolar diameters increased. Histological studies showed severe arteriolosclerosis and glomerulosclerosis in the DOCA rats. The results show that these arteriolar changes might contribute to the reduction of glomerular capillary pressure in the development of DOCA-salt hypertension. However, they are not sufficient to protect glomeruli from hypertensive damage.

Effects of sairei-to on the relapse of steroid-dependent nephrotic syndrome.

Sairei-To (Chai-Ling-Tang) was administered to four patients with steroid-dependent relapsing nephrotic syndrome. It was associated with prednisolone and immunosuppressive agents. Histological diagnosis was minimal change in three patients and mild focal glomerulonephritis in one patient. After the start of Sairei-To administration the relapse was markedly suppressed in three patients but not at all in the other. Although we could consider Sairei-To effective for steroid-dependent nephrotic syndrome in the present study, a larger study is necessary to confirm its efficacy.

Effects of a Japanese medicinal plant on the rat subtotal nephrectomy model: evaluation of its effect by microvascular casts.

Effects of a Japanese medicinal plant named Sairei-To, were examined in a rat experimental renal disease. Thirteen weeks after subtotal nephrectomy, the blood pressures in rats given Sairei-To (Sairei-To rats) were lower than those without Sairei-To. The urinary protein excretions and glomerular sclerosis were markedly decreased in the Sairei-To treated rats. Arteriolar diameters were measured using microvascular casts. The afferent and efferent arterioles were both significantly dilatated. The efferent arterioles in Sairei-To rats were dilated to a greater extent than that of the afferent. These results indicated that Sairei-To lessened renal damages in the rat subtotal nephrectomy model, possibly through the blood pressure reduction and the efferent arteriolar dilatation.

Variations in arterioles in spontaneously hypertensive rats. Morphometric analysis of afferent and efferent arterioles.

In the present study, the diameters of afferent and efferent arterioles of kidneys from spontaneously hypertensive rats (SHR) were evaluated and compared with those from Wistar Kyoto rats. (WKY) using a vascular cast model. At 4 weeks of age, the blood pressure was slightly higher in SHR than in WKY (124 +/- 1 vs 116 +/- 7 mmHg, ns). The diameters of afferent arterioles in SHR were smaller than those in WKY (10.3 +/- 0.6 vs 12.3 +/- 0.7 microns, P less than 0.001), whereas the diameters of efferent arterioles were comparable in the two strains. At 20 weeks of age, the blood pressure was markedly elevated in SHR than in WKY (192 +/- 5 vs 140 +/- 4 mmHg, P less than 0.001). The diameters of afferent arterioles in SHR at this age were much smaller than those in WKY (14.3 +/- 0.5 vs 17.1 +/- 0.6 microns, P less than 0.01). The diameters of efferent arterioles in SHR were, however, larger than those in WKY (15.4 +/- 1.2 vs 12.9 +/- 0.4 microns, P less than 0.05). The net effect of these changes in arteriolar size helps to maintain normal intraglomerular pressure and to protect glomeruli from damage due to hypertension.

Hexacosanoate contents in Japanese common foods.

Recently, a diet enriched in oleate and moderately restricted in hexacosanoate (C26:0) was found effective to reduce the plasma very long chain fatty acid (VLCFA) levels in patients with adrenoleukodystrophy (ALD), an X-linked disorder characterized by demyelination of the adrenal cortex and cerebral white matter, and accumulation of saturated VLCFA, particularly C26:0, in tissues of the demyelination. The information about the C26:0 content in Japanese food was, however, almost nil except for one report about foods in the USA, but this did not include some Japanese common foods. With the hope of treating an ALD patient in our hospital, C26:0 contents in Japanese common foods (42 items) were measured. In our case, a one-hour direct transesterification method was used to obtain methylesters of total fatty acids in foods and they were applied directly to a selected ion monitoring gas chromatography-mass spectrometry for the quantitative C26:0 analysis. The C26:0 content in nuts and seeds as well as in fats and oils was found to be significantly higher than in other foods; the content was highest in peanuts. The content in almost all kinds of examined fishes, the common protein foods in Japan, was relatively low. From these data and that in the national nutrition survey in 1986, the daily intake of C26:0 from the average Japanese diet could be estimated to be 12-36 mg. It can be recommended, therefore, that nuts and seeds as well as fats and oils should be restricted as severely as possible from the diet of ALD patients in Japan in order to keep daily C26:0 intake below 10 mg as recommended in the USA.

Interaction of secretory protein precursors with phospholipids in liposomes.

The precursors of secretory proteins were synthesized in a reticulocyte lysate system programmed with rat serum albumin or human placental lactogen mRNA and their interaction with phospholipids in liposomes was studied. The precursor proteins could bind to acidic phospholipids that have an exposed phosphate such as dicetyl phosphate and phosphatidic acid or a phosphate that is covered by a small moiety such as phosphatidylglycerol. The binding of precursor proteins was dependent on the mol% of acidic phospholipids in lecithin-liposomes, increased with elevation of temperature in the range of 0 to 45 degrees C, and was not inhibited by the addition of a large excess of mature proteins. Mature proteins or proalbumin showed no significant binding to the liposomes containing acidic phospholipids. About 15% of the acid-precipitable radioactivity bound to the liposomes was resistant to protease digestion. This radioactivity was shown to correspond to methionine-containing peptides with molecular weights of 2,500 to 3,500. These results indicate that the post-translational insertion of a small part of the precursor proteins into the membrane did occur with the present model system, but the post-translational transfer of precursor proteins across the membrane did not.

Colorimetric determination of glycine conjugates of bile acids.

A simple assay was developed for the determination of glycine-conjugated bile acids. Samples containing the glycine-conjugated bile acids in the range from 15 to 250 nmol were acidified with HCl, and extracted with ethyl acetate containing ethanol (50 ml/l). An aliquot of the organic phase was evaporated to dryness, and the dried residue was treated to develop the color by the addition of acetic anhydride, pyridine and a trace amount of phosphoric acid. the absorbance was measured at 429 or 456 nm after the reaction at 50 degrees C for 2 h. Color development did not occur with unconjugated and taurine-conjugated bile acid. Beer's law was obeyed from 12.5 to 200 nmol in a cuvette. The recovery of the conjugates from the rabbit gall bladder bile and liver homogenate was satisfactory. This method requires no hydrolysis step and is applicable to the determination of glycine-conjugated bile acids in bile, duodenal aspirate and liver homogenate.