Table 1. Effect of buffers and additives on the l-citrulline assay in the presence of 25 μm l-citrulline.

	l-citrulline μm	Color as % of control
Homogenization buffera
HB1	17.0±0.8	68
HB2	<Minimal	<1
HB3	24.9±0.6	100
HB4	>100b	464
Buffer base
1% Triton	13.0±0.9	52
1 m HEPES	27.5±0.4	110
0.3 m sucrose	>100	500
0.9% normal saline	24.6±0.6	98
0.1 m sodium phosphate	24.9±0.6	100
100 mm urea	>100	1419
Additives
0.1 m DTT	>100	384
1% 2-mercaptoethanol	<Minimal	<1
0.5% Tween	22.2±1.0	89
1% SDS	22.3±3.7	89
0.5 m EDTA	25.6±3.0	102
0.2 m EGTA	33.6±0.6	134
1 mm ADMA	22.2±0.8	89
Protease inhibitorsc	23.2±0.3	95
4% sulfosalicylic acid	28.2±1.0	113
Protein
1 mg/ml BSA	33.6±2.8b	134
2 mg/ml BSA	43.3±7.8b	173
1 mg/ml kidney homogenate	76.3±12.1b	305
2 mg/ml kidney homogenate	>100b	415
Protein with deproteinization
1 mg/ml BSA	26.1±0.8	107
2 mg/ml BSA	25.7±0.8	103
1 mg/ml kidney homogenate	63.4±1.1	253
2 mg/ml kidney homogenate	82.6±1.2	330

ADMA, asymmetric dimethylarginine; BSA, bovine serum albumin; EDTA, ethylene-diaminetetraacetic acid; EGTA, ethylene glycol-bis(â-aminoethyl ether)-N,N,N′,N′,-tetraacetic acid; HEPES, hydroxyethylpiperazine-N′-2-ethanesulfonic; SDS, sodium dodecyl sulfate; DTT, dl-dithiothreitol.

The values reflect effect of an additive on the color generated by a 25 μm l-citrulline standard (taken as 100%).

^aHB1, pH=6.8 contained 20 mm Tris, 1% Triton X-100, 5 mm EDTA, 10 mm EGTA, 2 mm DTT, 1 mm sodium orthovanadate, 0.1 mg/ml phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and aprotinin; HB2 contained 0.1 m sodium phosphate, pH=6.5 containing 2 mm 2-mercaptoethanol;¹⁰ HB3 contained 0.1 m sodium phosphate, pH=6.5;¹² and HB4 was RIPA buffer (Santa Cruz, CA, USA), containing 20 mm Tris, pH=7.6, 137 mm sodium chloride, 0.2% Nonidet P-40, 0.1% sodium deoxycholate, 0.02% SDS, 0.0008% sodium azide, and protease inhibitors.

^bThe supernatant was opalescent.

^cProtease inhibitors: 0.1 mg/ml phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and aprotinin.